Mammalian sweet taste receptors

ABSTRACT

The present invention provides isolated nucleic acid and amino acid sequences of sweet taste receptors comprising two heterologous G-protein coupled receptor polypeptides from the T 1 R family of sensory G-protein coupled receptors, antibodies to such receptors, methods of detecting such nucleic acids and receptors, and methods of screening for modulators of sweet taste receptors.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to U.S. Ser. No. 60/302,898,filed Jul. 3, 2001, herein incorporated by reference in its entirety.

The present application is related to U.S. Ser. No. 60/095,464, filedJuly.28, 1998; U.S. Ser. No. 60/112,747, filed Dec. 17, 1998; U.S. Ser.No. 09/361,631, filed Jul. 27, 1999; U.S. Ser. No. 60/094,465, and filedJul. 28, 1998; U.S. Ser. No. 09/361,652, filed Jul. 27, 1000, hereineach incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

Not applicable.

FIELD OF THE INVENTION

The sense of taste provides animals with valuable information about thequality and nutritional value of food. Previously, we identified a largefamily of mammalian taste receptors involved in bitter taste perception(the T2Rs). We now report the characterization of mammalian sweet tastereceptors. First, transgenic rescue experiments prove that the Sac locusencodes T1R3, a member of the T1R family of candidate taste receptors.Second, using a heterologous expression system, we demonstrate that T1R2and T1R3, when expressed in the same cell, function as a sweet receptor,recognizing sweet tasting molecules as diverse as sucrose, saccharin,dulcin and acesulfame-K. The T1R family therefore forms sweet receptorscomprising polypeptides such as T1R2 and T1R3, and T1R1 and T1R3.Finally, we present a detailed analysis of the patterns of expression ofT1Rs and T2Rs, thus providing a view of the representation of sweet andbitter taste at the periphery.

BACKGROUND OF THE INVENTION

Our sense of taste is capable of detecting and responding to sweet,bitter, sour, salty and umami stimuli (reviewed by Lindemann, 1996). Itis also responsible for distinguishing between these various tastemodalities, for instance, the sweetness of honey from the bitterness oftonic water; the sourness of unripe fruit from the saltiness of theocean. This discriminatory power provides valuable sensory input: bitterreceptors elicit aversive behavioral reactions to noxious substances,while sweet receptors allow recognition of high caloric food sources.

We have been interested in basic questions of taste signal detection andinformation coding, and have focused on the isolation andcharacterization of genes encoding sweet and bitter taste receptors. Theidentification of taste receptors generates powerful molecular tools toinvestigate not only the function of taste receptor cells, but also thelogic of taste coding. For example, defining the size and diversity ofthe receptor repertoire provide evidence for how a large number ofchemosensory ligands may be recognized (i.e., molecular diversity),while analysis of the patterns of receptor expression contributesimportant insight to our understanding of chemosensory discriminationand coding. Recently, we described the isolation of two novel familiesof G-protein coupled receptors (GPCRs) expressed in subsets of tastereceptor cells of the tongue and palate (T1Rs and T2Rs; Hoon et al.,1999; Adler et al., 2000). One of these, the T2Rs, is a family of ˜30different genes that include several functionally validated mammalianbitter taste receptors (Adler et al., 2000; Chandrashekar et al., 2000;Matsunami et al., 2000). Nearly all of the T2R-genes are clustered inregions of the genome that have been genetically implicated incontrolling responses to diverse bitter tastants in humans and mice,consistent with their proposed role as bitter taste receptors (Adler etal., 2000).

Notably, most T2Rs are co-expressed in the same subset of taste receptorcells (Adler et al., 2000), suggesting that these cells are capable ofresponding to a broad array of bitter compounds, but not discriminatingbetween them. This is logical for a sensory modality like bitter, inwhich the animal needs to recognize and react to many noxious tastants,but not necessarily discriminate between them (i.e., we need to knowthat a tastant is bad news, but not necessarily what makes it bad). Thisinterpretation is consistent with behavioral and psychophysical findingsin rodents and humans demonstrating limited discrimination betweenvarious bitter tastants (McBurney and Gent, 1979).

How is sweet taste specified? There is considerable evidence thatG-protein coupled receptors are also involved in this taste modality(Lindemann, 1996). In contrast to bitter taste, the number ofbiologically relevant sweet tastants is modest. Thus, we might expectthe sweet receptor family to be quite small. Interestingly,psychophysical, behavioral and electrophysiological studies suggest thatanimals distinguish between various sweet tastants (Schiffman et al.,1981; Ninomiya et al., 1984; Ninomiya et al., 1997), perhaps reflecting(and predicting) the organization of the sweet taste system intodistinct types of sweet receptor cells and pathways.

Genetic studies of sweet tasting have identified a single principallocus in mice influencing responses to several sweet substances (Fuller,1974; Lush, 1989). This locus, named Sac, determines thresholddifferences in the ability of some strains to distinguishsaccharin-containing solutions from water (Fuller, 1974). Sac tastersrespond to ˜5-fold lower concentrations of saccharin than“sweet-insensitive” Sac non-taster mice (Fuller, 1974; Capeless andWhitney, 1995); additionally, Sac influences preferences to sucrose,acesulfame-K and dulcin (Lush, 1989). Recently, several groups reportedthat a T1R-related gene, T1R3, might encode Sac (Kitagawa et al., 2001;Max et al., 2001; Montmayeur et al., 2001; Sainz et al., 2001).

We now demonstrate that transgenic expression of T1R3 from a tasterstrain transforms sweet-insensitive animals to tasters, affirming T1R3as the Sac gene. We then developed a cell-based reporter system to provethat T1Rs encode functional sweet taste receptors. Lastly, we show thatthe patterns of T1R expression define at least three distinct celltypes, and that sweet and bitter receptors are tightly segregated at theperiphery.

BRIEF SUMMARY OF THE INVENTION

The present invention thus provides for the first time a sweet tastereceptor comprising a T1R3 polypeptide. The present invention providessweet taste receptor comprising a T1R3 polypeptide and a heterologousmember of the T1R family, e.g., T1R1 or T1R2, that transduces a signalin response to sweet taste ligands when T1R3 and either T1R1 or T1R2 areco-expressed in the same cell. In one embodiment, the T1R3 polypeptideand the heterologous T1R polypeptide form a heterodimer. In anotherembodiment, the T1R3 polypeptide and the heterologous T1R polypeptideare non-covalently linked. In another embodiment, the T1R3 polypeptideand the heterologous T1R polypeptide are covalently linked.

In one aspect, the present invention provides an sweet taste receptorcomprising a T1R3 polypeptide, the T1R3 polypeptide comprising greaterthan about 70% amino acid sequence identity to an amino acid sequence ofSEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:25, or encoded bya nucleotide sequence hybridizing under moderately or highly stringenthybridization conditions to a nucleotide sequence encoding an amino acidsequence of SEQ ID NO: 15, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:25.

In one embodiment, the receptor specifically binds to polyclonalantibodies generated against SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23,or SEQ ID NO:25. In another embodiment, the receptor has G-proteincoupled receptor activity. In another embodiment, the T1R3 polypeptidehas an amino acid sequence of SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23,or SEQ ID NO:25. In another embodiment, the receptor is from a human, arat, or a mouse.

In one aspect, the present invention provides a sweet taste receptor,the receptor comprising a T1R3 polypeptide and a heterologouspolypeptide, the T1R3 polypeptide comprising greater than about 70%amino acid sequence identity to an amino acid sequence of SEQ ID NO:15,SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:25, or encoded by a nucleotidesequence hybridizing under moderately or highly stringent hybridizationconditions to a nucleotide sequence encoding an amino acid sequence ofSEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:25.

In one embodiment, the heterologous polypeptide is a T1R family member.In another embodiment, the heterologous polypeptide is T1R1 or T1R2. Inone embodiment, the T1R1 polypeptide is encoded by a nucleotide sequencethat hybridizes under moderately or highly stringent conditions to anucleotide sequence encoding SEQ ID NO:1, 2, or 3. In one embodiment,the T1R2 polypeptide is encoded by a nucleotide sequence that hybridizesunder moderately or highly stringent conditions to a nucleotide sequenceencoding SEQ ID NO:7, 8, or 9. In one embodiment, the receptorspecifically binds to polyclonal antibodies generated against SEQ IDNO:15, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:25. In anotherembodiment, the receptor has G-protein coupled receptor activity. Inanother embodiment, the T1R3 polypeptide has an amino acid sequence ofSEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:25. In anotherembodiment, the receptor is from a human, a rat, or a mouse.

In one embodiment, the T1R3 polypeptide and the T1R polypeptide form aheterodimer. In one embodiment, the T1R3 polypeptide and the T1Rheterologous polypeptide are non-covalently linked. In anotherembodiment, the T1R3 polypeptide and the T1R heterologous polypeptideare covalently linked.

In one aspect, the present invention provides an isolated polypeptidecomprising an extracellular, a transmembrane domain, or a cytoplasmicdomain of a sweet taste receptor, the extracellular, a transmembranedomain, or a cytoplasmic domain comprising greater than about 70% aminoacid sequence identity to the extracellular, a transmembrane domain, ora cytoplasmic domain of SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, or SEQID NO:25. In another embodiment, the extracellular, transmembrane, orcytoplasmic domain hybridize under moderately or highly stringentconditions to an extracellular, transmembrane, or cytoplasmic domain ofSEQ ID NO:15, 20, 23, or 25.

In one embodiment, the polypeptide encodes the extracellular, atransmembrane domain, or a cytoplasmic domain of SEQ ID NO:15, SEQ IDNO:20, SEQ ID NO:23, or SEQ ID NO:25. In another embodiment, theextracellular, a transmembrane domain, or a cytoplasmic domain iscovalently linked to a heterologous polypeptide, forming a chimericpolypeptide. In another embodiment, the chimeric polypeptide hasG-protein coupled receptor activity.

In one aspect, the present invention provides an antibody thatselectively binds to a sweet taste receptor, the receptor comprising aT1R3 polypeptide and a heterologous polypeptide, the antibody raisedagainst a receptor comprising a T1R3 polypeptide comprising greater thanabout 70% amino acid sequence identity to an amino acid sequence of SEQID NO:15, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:25, or encoded by anucleotide sequence hybridizing under highly or moderately stringenthybridization conditions to a nucleotide sequence encoding an amino acidsequence of SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:25.In one embodiment, the T1R3 polypeptide forms a heterodimeric receptor,either by covalent or non-covalent linkage, with a T1R polypeptide, towhich the antibody specifically binds.

In another aspect, the present invention provides a method foridentifying a compound that modulates sweet taste signaling in tastecells, the method comprising the steps of: (i) contacting the compoundwith a sweet receptor comprising a T1R3 polypeptide, the polypeptidecomprising greater than about 70% amino acid sequence identity to theextracellular domain of SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, or SEQID NO:25; or encoded by a nucleotide sequence hybridizing undermoderately or highly stringent hybridization conditions to a nucleotidesequence encoding an amino acid sequence of SEQ ID NO:15, SEQ ID NO:20,SEQ ID NO:23, or SEQ ID NO:25; and (ii) determining the functionaleffect of the compound upon the receptor.

In one embodiment, the receptor comprises T1R3, is heterodimericreceptor, and is linked to a heterologous polypeptide either covalentlyor non-covalently. In another embodiment, the receptor comprises T1R1and T1R3. In another embodiment, the receptor comprises T1R2 and T1R3.In another embodiment, the polypeptide has G-protein coupled receptoractivity. In another embodiment, the functional effect is determined invitro. In another embodiment, the receptor is linked to a solid phase,either covalently or non-covalently. In another embodiment, thefunctional effect is determined by measuring changes in intracellularcAMP, IP3, or Ca2+. In another embodiment, the functional effect is achemical or phenotypic effect. In another embodiment, the functionaleffect is a physical effect. In another embodiment, the functionaleffect is determined by measuring binding of the compound to theextracellular domain of the receptor. In another embodiment, thepolypeptide is recombinant. In another embodiment, the polypeptide isexpressed in a cell or cell membrane. In another embodiment, the cell isa eukaryotic cell, e.g., a mammalian cell, e.g., a human cell.

In one aspect, the present invention provides an isolated nucleic acidencoding a T1R3 polypeptide, the polypeptide comprising greater thanabout 70% amino acid identity to an amino acid sequence of SEQ ID NO:15,SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:25.

In one embodiment, the nucleic acid comprises a nucleotide sequence ofSEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:22, or SEQ ID NO:24. In anotherembodiment, the nucleic acid is amplified by primers that selectivelyhybridize under stringent hybridization conditions to SEQ ID NO:14, SEQID NO:19, SEQ ID NO:22, or SEQ ID NO:24.

In another aspect, the present invention provides an isolated nucleicacid encoding a T1R3 polypeptide, wherein the nucleic acid specificallyhybridizes under moderately or highly stringent conditions to a nucleicacid encoding SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:25or having the sequence of SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:22, orSEQ ID NO:24.

In another aspect, the present invention provides an isolated nucleicacid encoding a T1R3 polypeptide, the polypeptide comprising greaterthan about 70% amino acid identity to a polypeptide having a sequence ofSEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:25, wherein thenucleic acid selectively hybridizes under moderately stringenthybridization conditions to a nucleotide sequence of SEQ ID NO:14, SEQID NO:19, SEQ ID NO:22, or SEQ ID NO:24.

In another aspect, the present invention provides an isolated nucleicacid encoding an extracellular domain, a transmembrane domain, or acytoplasmic domain of a T1R3 polypeptide, the extracellular domain, atransmembrane domain, or a cytoplasmic domain having greater than about70% amino acid sequence identity to the extracellular domain, atransmembrane domain, or a cytoplasmic domain of SEQ ID NO:15, SEQ IDNO:20, SEQ ID NO:23, or SEQ ID NO:25.

In another aspect, the present invention provides an expression vectorcomprising a nucleic acid encoding a polypeptide comprising greater thanabout 70% amino acid sequence identity to an amino acid sequence of SEQID NO:15, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:25. In anotheraspect, the present invention provides a host cell transfected with theexpression vector.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. T1R3 maps to the Sac locus

(a) Radiation hybrid and STS mapping localized all three T1R genes tothe distal end of chromosome 4. The T1R3 gene is closely linked toD18346, an STS marker within the Sac genetic interval (Kitagawa et al.,2001; Li et al., 2001; Max et al., 2001; Montmayeur et al., 2001; Sainzet al., 2001). (b) Cladogram showing sequence similarity between human(h) and mouse (m) T1Rs and related receptors (Nakanishi, 1992; Brown etal., 1993; Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba andTirindelli, 1997; Kaupmann et al., 1997; Hoon et al., 1999); mouse V2Rsdo not have human counterparts.

FIG. 2. T1R3 encodes Sac

(a) Schematic diagram indicating structure of the T1R3 gene andtransgenic construct. The alternate 3′-UTRs used for genotyping and insitu hybridization are highlighted in green and red. (b) In situhybridization demonstrated perfect concordance in the expression patternof the T1R3 transgene (red) and the endogenous gene (green). The dottedlines illustrate the outline of selected taste buds; sections were cutperpendicular to the planes shown in FIG. 3.(c-h) Taste preferences ofcontrol and transgenic animals (solid red circles) were measured usingstandard two bottle preference tests. The behavioral responses of miceexpressing the T1R3 transgene to saccharin and sucrose (panels c and d)were indistinguishable from those of the control taster mice (C57BL/6;open red circles). Siblings without the transgene (solid black circles)behaved like 129/Sv non-taster control mice (open black circles).Responses to bitter, salty, sour and umami stimuli (panels e-h) were notaffected by presence of the transgene.

FIG. 3. Expression of T1Rs in subsets of taste receptor cells In situhybridizations with digoxigenin-labeled antisense RNA probesdemonstrated that T1R3 is expressed in subsets of mouse taste receptorcells (upper panels). Approx. 30% of cells in fungiform, circumvallate,foliate and palate taste buds express T1R3. Shown for comparison aresimilar, but not serial, sections labeled with T1R1 and T1R2 (middle andlower panels; see also Hoon et al., 1999 and FIG. 4). The dotted linesillustrate the outline of a sample taste bud. Note that the selectivityof T1R3 expression closely resembles that of T1R1 plus T1R2.

FIG. 4. T1R expression patterns define three cell types

Double-label fluorescent in situ hybridization was used to directlyexamine the overlap in cellular expression of T1Rs. Two-channelfluorescent images (1-2 μm optical sections) are overlaid on differenceinterference contrast images. (a) Fungiform papillae illustratingco-expression of T1R1 (red) and T1R3 (green). At least 90% of the cellsexpressing T1R1 also express T1R3; similar results were observed in thepalate. Note the presence of some T1R3 positive but T1R1 negative cells.(b) Circumvallate papillae illustrating co-expression of T1R2 (green)and T1R3 (red). Every T1R2 positive cell expresses T1R3.

FIG. 5. T1R2+3 responds to sweet tastants

HEK-293 cells co-expressing promiscuous G proteins and rat T1R2 and T1R3were stimulated with various sweet compounds. Robust increases in[Ca²⁺]i were observed upon addition of 250 mM sucrose (d, g), 180 μMGA-2 (e, h) and 10 mM acesulfame-K (f, i). Panels a-c show cells priorto stimulation. No responses were detected without receptors (panel j)or promiscuous G proteins (panel k). Glucose and several other sweettastants (see next figure) did not activate this receptor combination(panel 1); scales indicate [Ca²⁺]i (nM) determined from FURA-2 F₃₄₀/F₃₈₀ratios. Line traces (g-i) show the kinetics of the [Ca²⁺]i changes forrepresentative cells from panels (d-f). The bar indicates the time andduration of the stimulus.

FIG. 6. T1R2+3 selectively responds to a broad range of sweet compounds

(a) The responses of the T1R2+3 receptor combination were specific tosucrose, fructose and five artificial sweeteners. Concentrations usedare: GA-1 (500 μM); GA-2 (500 μM); sucrose (250 mM); fructose (250 mM),acesulfame-K (10 mM); dulcin (2 mM), sodium saccharin (5 mM); N-methylsaccharin (5 mM), glucose (250 mM); maltose (250 mM); lactose (250 mM);galactose (250 mM); palatinose (250 mM); thaumatin (0.1%); sodiumcyclamate (15 mM); aspartame (2mM). Columns represent the mean±SEM of aminimum of 16 independent determinations. (b) Dose response of T1R2+3 tosucrose, saccharin, acesulfame-K and GA-2. The relative changes in[Ca²⁺]i are shown as FURA-2 (F₃₄₀/F₃₈₀) ratios normalized to theresponses obtained for the highest concentration of each compound. Eachpoint represents the mean±SEM of a minimum of 20 assays. (c) Kineticsand desensitization of T1R2+3 sweet responses. Cells expressing T1R2+3were stimulated with multiple pulses of sweet tastants; GA-2 (360 μM),sucrose, (suc: 250 mM), acesulfame-K (ace: 10 mM), cyclamate (sic: 15mM), glucose (glu: 250 mM) and aspartame (asp: 2 mM). Dots andhorizontal bars indicate the time and duration of the stimulus. Sucrose,GA-2 and acesulfame-K elicit robust responses; repeated or prolongedstimulation with any one of these tastants (e.g., GA-2) results in adecreased response indicative of desensitization. Stimulation withsucrose or acesulfame-K immediately after GA-2 results in an attenuatedresponse suggesting cross-desensitization. The trace was derived from 80responding cells in the field of view.

FIG. 7. T1Rs and T2Rs are segregated in distinct populations of tastereceptor cells

Double-label fluorescent in situ hybridization was used to examine thedegree of overlap between the T1R and T2R families of sweet and bittertaste receptors. (a) T1R3 (green) and T2Rs (a mixture of 20 receptors,red) are never co-expressed. (b) A section through a circumvallatepapilla is shown (b) as in panel (a), but with a mixture of all threeT1Rs (green) versus twenty T2Rs in a foliate papilla.

DETAILED DESCRIPTION

Introduction

The present invention provides sweet taste receptors comprising membersof the T1R family of G-protein coupled receptors. In a preferredembodiment, the present invention provides sweet taste receptorcomprising a T1R3 polypeptide and a second, heterologous T1Rpolypeptide, e.g., T1R1 or T1R2. These sweet taste receptors are GPCRcomponents of the taste transduction pathway, and when co-expressed inthe same cell, the polypeptides transduce signal in response to sweettaste ligand.

These nucleic acids and proteins encoding the receptors provide valuableprobes for the identification of taste cells, as the nucleic acids arespecifically expressed in taste cells. For example, probes for GPCRpolypeptides and proteins can be used to identity subsets of taste cellssuch as foliate cells, palate cells, and circumvallate cells, orspecific taste receptor cells, e.g., sweet taste receptor cells. Asdescribed below, T1R1 and T1R3, and T1R2 and T1R3 are co-expressed inspecific taste receptor cell subsets. They also serve as tools for thegeneration of taste topographic maps that elucidate the relationshipbetween the taste cells of the tongue and taste sensory neurons leadingto taste centers in the brain. Furthermore, the nucleic acids and theproteins they encode can be used as probes to dissect taste-inducedbehaviors.

The invention also provides methods of screening for modulators, e.g.,activators, inhibitors, stimulators, enhancers, agonists, andantagonists, of these novel sweet taste receptors comprising T1R3 andanother member of the T1R family such as T1R1 or T1R2. Such modulatorsof sweet taste transduction are useful for pharmacological and geneticmodulation of sweet taste signaling pathways, and for the discovery ofnovel sweet taste ligands. These methods of screening can be used toidentify high affinity agonists and antagonists of sweet taste cellactivity. These modulatory compounds can then be used in the food andpharmaceutical industries to customize taste. Thus, the inventionprovides assays for taste modulation, where the T1R3-comprising receptoracts as an direct or indirect reporter molecule for the effect ofmodulators on sweet taste transduction. GPCRs can be used in assays,e.g., to measure changes in ligand binding, G-protein binding,regulatory molecule binding, ion concentration, membrane potential,current flow, ion flux, transcription, signal transduction,receptor-ligand interactions, neurotransmitter and hormone release; andsecond messenger concentrations, in vitro, in vivo, and ex vivo. In oneembodiment, a receptor comprising T1R3 can be used as an indirectreporter via attachment to a second reporter molecule such as greenfluorescent protein (see, e.g., Mistili & Spector, Nature Biotechnology15: 961-964 (1997)). In another embodiment, a receptor comprising T1R3is recombinantly expressed in cells, and modulation of tastetransduction via GPCR activity is assayed by measuring changes in Ca2+levels.

Methods of assaying for modulators of taste transduction include invitro ligand binding assays using receptors comprising T1R3, portionsthereof such as the extracellular domain, or chimeric proteinscomprising one or more domains of T1R3, and in in vivo (cell-based andanimal) assays such as oocyte T1R3 receptor expression; tissue culturecell T1R3 receptor expression; transcriptional activation of T1R3;phosphorylation and dephosphorylation of GPCRs; G-protein binding toGPCRs; ligand binding assays; voltage, membrane potential andconductance changes; ion flux assays; changes in intracellular secondmessengers such as cAMP and inositol triphosphate; changes inintracellular calcium levels; and neurotransmitter release.

DEFINITIONS

A “T1R family taste receptor” refers to a receptor comprising a memberof the T1R family of G-protein coupled receptors, e.g., T1R1, T1R2, andT1R3, or any combination thereof. In one embodiment, the T1R familyreceptor comprises T1R3 (a “T1R3-comprising taste receptor” or a“T1R3-comprising sweet taste receptor”) and a heterologous polypeptideof the T1R family. In one embodiment, the receptor comprises T1R1 andT1R3. In another embodiment, the receptor comprises T1R2 and T1R3. Inone embodiment the T1R3-comprising receptor is active when the twomembers of the receptor are co-expressed in the same cell, e.g., T1R1and T1R3 or T1R2 and T1R3. In another embodiment, the T1R polypeptidesare co-expressed in the same cell and form a heterodimeric receptor, inwhich the T1R polypeptides of the receptor are non-covalently linked orcovalently linked. The receptor has the ability to recognize a sweettasting molecule such as sucrose, saccharin, dulcin, acesulfame-K, aswell as other molecules, sweet and non-sweet, as described herein. Thesemolecules are examples of compounds that “modulate sweet taste signaltransduction” by acting as ligands for the sweet G protein coupledreceptor comprising T1R3.

The terms “GPCR-B3 or T1R1,” “GPCR-B4 or T1R2,” and “T1R3” or a nucleicacid encoding “GPCR-B3 or T1R1,” “GPCR-B4 or T1R2,” and “T1R3” refer tonucleic acid and polypeptide polymorphic variants, alleles, mutants, andinterspecies homologs that are members of the T1R family of G proteincoupled receptors and: (1) have an amino acid sequence that has greaterthan about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%,90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greateramino acid sequence identity, preferably over a region of over a regionof at least about 25, 50, 100, 200, 500, 1000, or more amino acids, toan amino acid sequence encoded by SEQ ID NO:1, 2, 3, 7, 8, 9, 15, 18,20, 23, or 25; (2) bind to antibodies, e.g., polyclonal antibodies,raised against an immunogen comprising an amino acid sequence encoded bySEQ ID NO:1, 2, 3, 7, 8, 9, 15, 18, 20, 23, or 25, and conservativelymodified variants thereof; (3) specifically hybridize under stringenthybridization conditions to an anti-sense strand corresponding to anucleic acid sequence encoding a T1R protein, e.g., SEQ ID NO:4, 5, 6,10, 11, 12, 13, 14, 16, 17, 19, 21, 22, or 24, and conservativelymodified variants thereof; (4) have a nucleic acid sequence that hasgreater than about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%,preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or highernucleotide sequence identity, preferably over a region of at least about25, 50, 100, 200, 500, 1000, or more nucleotides, to SEQ ID NO:4, 5, 6,10, 11, 12, 13, 14, 16, 17, 19, 21, 22, or 24. The T1R familypolypeptide ofthe invention (e.g., T1R1 , T1R2, or T1R3) orT1R3-comprising receptor further has G protein coupled receptoractivity, either alone or when co-expressed in the same cell, or whenco-expressed as a heterodimer with another T1R family member. Accessionnumbers for amino acid sequences and nucleotide sequences of T1R1, T1R2,and T1R3 can be found in GenBank.

T1R proteins have “G-protein coupled receptor activity,” e.g., they bindto G-proteins in response to extracellular stimuli, such as ligandbinding, and promote production of second messengers such as IP3, cAMP,and Ca2+ via stimulation of enzymes such as phospholipase C andadenylate cyclase. Such activity can be measured in a heterologous cell,by coupling a GPCR (or a chimeric GPCR) to either a G-protein orpromiscuous G-protein such as Gα15, and an enzyme such as PLC, andmeasuring increases in intracellular calcium using (Offermans & Simon,J. Biol. Chem. 270: 15175-15180 (1995)). Receptor activity can beeffectively measured, e.g., by recording ligand-induced changes in[Ca²⁺]i using fluorescent Ca²⁺-indicator dyes and fluorometric imaging.

Such GPCRs have transmembrane, extracellular and cytoplasmic domainsthat can be structurally identified using methods known to those ofskill in the art, such as sequence analysis programs that identifyhydrophobic and hydrophilic domains (see, e.g., Kyte & Doolittle, J.Mol. Biol. 157: 105-132 (1982)). Such domains are useful for makingchimeric proteins and for in vitro assays of the invention (see, e.g.,WO 94/05695 and U.S. Pat. No. 5,508,384).

The phrase “functional effects” in the context of assays for testingcompounds that modulate activity (e.g., signal transduction) of a sweettaste receptor or protein of the invention includes the determination ofa parameter that is indirectly or directly under the influence of a GPCRor sweet taste receptor, e.g., a physical, phenotypic, or chemicaleffect, such as the ability to transduce a cellular signal in responseto external stimuli such as ligand binding, or the ability to bind aligand. It includes binding activity and signal transduction.“Functional effects” include in vitro, in vivo, and ex vivo activities.

By “determining the functional effect” is meant assaying for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of a T1R GPCR protein or a sweet taste receptorcomprising one or more T1R GPCR proteins, e.g., physical (direct) andchemical or phenotypic (indirect) effects. Such functional effects canbe measured by any means known to those skilled in the art, e.g.,changes in spectroscopic characteristics (e.g., fluorescence,absorbance, refractive index); hydrodynamic (e.g., shape);chromatographic; or solubility properties for the protein; measuringinducible markers or transcriptional activation of the protein;measuring binding activity or binding assays, e.g., binding toantibodies; measuring changes in ligand binding activity or analogsthereof, either naturally occurring or synthetic; measuring cellularproliferation; measuring cell surface marker expression, measurement ofchanges in protein levels for T1R-associated sequences; measurement ofRNA stability; G-protein binding; GPCR phosphorylation ordephosphorylation; signal transduction, e.g., receptor-ligandinteractions, second messenger concentrations (e.g., cAMP, cGMP, IP3,PI, or intracellular Ca²⁺); neurotransmitter release; hormone release;voltage, membrane potential and conductance changes; ion flux;regulatory molecule binding; identification of downstream or reportergene expression (CAT, luciferase, β-gal, GFP and the like), e.g., viachemiluminescence, fluorescence, colorimetric reactions, antibodybinding, and inducible markers.

“Inhibitors,” “activators,” and “modulators” of T1R familypolynucleotide and polypeptide sequences and T1R family sweet receptorsare used to refer to activating, inhibitory, or modulating moleculesidentified using in vitro and in vivo assays of T1R polynucleotide andpolypeptide sequences and T1R family sweet receptors, includingheterodimeric receptors. Inhibitors are compounds that, e.g., bind to,partially or totally block activity, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate the activity orexpression of T1R family sweet receptors such as a receptor comprising aT1R3 polypeptide, e.g., antagonists. “Activators” are compounds thatincrease, open, activate, facilitate, enhance activation, sensitize,agonize, or up regulate a T1R family sweet receptor, such as a receptorcomprising a T1R3 polypeptide, e.g., agonists. Inhibitors, activators,or modulators also include genetically modified versions of T1R familysweet receptors, e.g., versions with altered activity, as well asnaturally occurring and synthetic ligands, antagonists, agonists,antibodies, antisense molecules, ribozymes, small chemical molecules andthe like. Such assays for inhibitors and activators include, e.g.,expressing T1R family sweet receptors in vitro, in cells, or cellmembranes, applying putative modulator compounds, and then determiningthe functional effects on activity, as described above. The sweet tastereceptor comprising a T1R3 polypeptide has the ability to recognize asweet tasting molecule such as sucrose, saccharin, dulcin, acesulfame-K,and other molecules, as described herein. These molecules are examplesof compounds that modulate sweet taste signal transduction by acting asextracellular ligands for the sweet G protein coupled receptor andactivating the receptor. In other embodiments, compounds that modulatesweet taste signal transduction are molecules that act as intracellularligands of the receptor, or inhibit or activate binding of anextracellular ligand, or inhibit or activate binding of intracellularligands of the receptor.

Samples or assays comprising T1R family sweet receptors that are treatedwith a potential activator, inhibitor, or modulator are compared tocontrol samples without the inhibitor, activator, or modulator toexamine the extent of inhibition. Control samples (untreated withinhibitors) are assigned a relative protein activity value of 100%.Inhibition of a T1R family sweet receptor is achieved when the activityvalue relative to the control is about 80%, preferably 50%, morepreferably 25-0%. Activation of a T1R family sweet receptor is achievedwhen the activity value relative to the control (untreated withactivators) is 110%, more preferably 150%, more preferably 200-500%(i.e., two to five fold higher relative to the control), more preferably1000-3000% higher.

The term “test compound” or “drug candidate” or “modulator” orgrammatical equivalents as used herein describes any molecule, eithernaturally occurring or synthetic, e.g., protein, oligopeptide (e.g.,from about 5 to about 25 amino acids in length, preferably from about 10to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 aminoacids in length), small organic molecule, polysaccharide, lipid (e.g., asphingolipid), fatty acid, polynucleotide, oligonucleotide, etc., to betested for the capacity to directly or indirectly modulation taste. Thetest compound can be in the form of a library of test compounds, such asa combinatorial or randomized library that provides a sufficient rangeof diversity. Test compounds are optionally linked to a fusion partner,e.g., targeting compounds, rescue compounds, dimerization compounds,stabilizing compounds, addressable compounds, and other functionalmoieties. Conventionally, new chemical entities with useful propertiesare generated by identifying a test compound (called a “lead compound”)with some desirable property or activity, e.g., inhibiting activity,creating variants of the lead compound, and evaluating the property andactivity of those variant compounds. Often, high throughput screening(HTS) methods are employed for such an analysis.

A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 daltons and less than about 2500 daltons, preferably lessthan about 2000 daltons, preferably between about 100 to about 1000daltons, more preferably between about 200 to about 500 daltons.

“Biological sample” include sections of tissues such as biopsy andautopsy samples, and frozen sections taken for histologic purposes. Suchsamples include blood, sputum, tissue, cultured cells, e.g., primarycultures, explants, and transformed cells, stool, urine, etc. Abiological sample is typically obtained from a eukaryotic organism, mostpreferably a mammal such as a primate e.g., chimpanzee or human; cow;dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird;reptile; or fish.

A “heterodimer” is a dimer comprising two different molecules, e.g., twodifferent polypeptides, where the molecules are associated via eithercovalent, e.g., through a linker or a chemical bond, or non-covalent,e.g., ionic, van der Waals, electrostatic, or hydrogen bonds linkages.The T1R2-comprising receptors of the invention function whenco-expressed in the same cell, or optionally when co-expressed so thatthey form a heterodimer, either covalently or non-covalently linked.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region (e.g., nucleotide sequences SEQ ID NO:1-25), whencompared and aligned for maximum correspondence over a comparison windowor designated region) as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection (see, e.g., NCBI web site or thelike). Such sequences are then said to be “substantially identical.”This definition also refers to, or may be applied to, the compliment ofa test sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions. Asdescribed below, the preferred algorithms can account for gaps and thelike. Preferably, identity exists over a region that is at least about25 amino acids or nucleotides in length, or more preferably over aregion that is 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85: 2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25: 3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with theparameters described herein, to determine percent sequence identity forthe nucleic acids and proteins of the invention. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains, e.g., extracellulardomains, transmembrane domains, and cytoplasmic domains. Domains areportions of a polypeptide that form a compact unit of the polypeptideand are typically 15 to 350 amino acids long. Typical domains are madeup of sections of lesser organization such as stretches of β-sheet andα-helices. “Tertiary structure” refers to the complete three dimensionalstructure of a polypeptide monomer. “Quaternary structure” refers to thethree dimensional structure formed by the noncovalent association ofindependent tertiary units. Anisotropic terms are also known as energyterms.

A particular nucleic acid sequence also implicitly encompasses “splicevariants.” Similarly, a particular protein encoded by a nucleic acidimplicitly encompasses any protein encoded by a splice variant of thatnucleic acid. “Splice variants,” as the name suggests, are products ofalternative splicing of a gene. After transcription, an initial nucleicacid transcript may be spliced such that different (alternate) nucleicacid splice products encode different polypeptides. Mechanisms for theproduction of splice variants vary, but include alternate splicing ofexons. Alternate polypeptides derived from the same nucleic acid byread-through transcription are also encompassed by this definition. Anyproducts of a splicing reaction, including recombinant forms of thesplice products, are included in this definition.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins whichcan be made detectable, e.g., by incorporating a radiolabel into thepeptide or used to detect antibodies specifically reactive with thepeptide.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology, ed. Ausubel, et al.

For PCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealingphase lasting 30 sec.-2 min., and an extension phase of about 72° C. for1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al., PCRProtocols, A Guide to Methods and Applications (1990).

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.Typically, the antigen-binding region of an antibody will be mostcritical in specificity and affinity of binding.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348: 552-554(1990))

For preparation of antibodies, e.g., recombinant, monoclonal, orpolyclonal antibodies, many technique known in the art can be used (see,e.g., Kohler & Milstein, Nature 256: 495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan,Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, ALaboratory Manual (1988); and Goding, Monoclonal Antibodies: Principlesand Practice (2d ed. 1986)). Techniques for the production of singlechain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produceantibodies to polypeptides of this invention. Also, transgenic mice, orother organisms such as other mammals, may be used to express humanizedantibodies. Alternatively, phage display technology can be used toidentify antibodies and heteromeric Fab fragments that specifically bindto selected antigens (see, e.g., McCafferty et al., Nature 348: 552-554(1990); Marks et al., Biotechnology 10: 779-783 (1992)).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

In one embodiment, the antibody is conjugated to an “effector” moiety.The effector moiety can be any number of molecules, including labelingmoieties such as radioactive labels or fluorescent labels, or can be atherapeutic moiety. In one aspect the antibody modulates the activity ofthe protein.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, often in a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular protein atleast two times the background and more typically more than 10 to 100times background. Specific binding to an antibody under such conditionsrequires an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies raised to a T1Rprotein or a heterodimeric T1R3-comprising sweet taste receptorcomprising a sequence of or encoded by SEQ ID NO:1-25, polymorphicvariants, alleles, orthologs, and conservatively modified variants, orsplice variants, or portions thereof, can be selected to obtain onlythose polyclonal antibodies that are specifically immunoreactive withT1R proteins and/or heterodimeric T1R3-comprising sweet taste receptorsand not with other proteins. In one embodiment, the antibodies reactwith a heterodimeric T1R3-comprising taste receptor, but not withindividual protein members of the T1R family. This selection may beachieved by subtracting out antibodies that cross-react with othermolecules. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988) for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity).

Isolation of Nucleic Acids Encoding T1R Family Members

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

T1R nucleic acids, polymorphic variants, orthologs, and alleles that aresubstantially identical to an amino acid sequences disclosed herein canbe isolated using T1R nucleic acid probes and oligonucleotides understringent hybridization conditions, by screening libraries.Alternatively, expression libraries can be used to clone T1R protein,polymorphic variants, orthologs, and alleles by detecting expressedhomologs immunologically with antisera or purified antibodies madeagainst human T1R or portions thereof.

To make a cDNA library, one should choose a source that is rich in T1RRNA, e.g., taste buds such as circumvallate, foliate, fungiform, andpalate. The mRNA is then made into cDNA using reverse transcriptase,ligated into a recombinant vector, and transfected into a recombinanthost for propagation, screening and cloning. Methods for making andscreening cDNA libraries are well known (see, e.g., Gubler & Hoffman,Gene 25: 263-269 (1983); Sambrook et al., supra; Ausubel et al., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196: 180-182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72: 3961-3965 (1975).

An alternative method of isolating T1R nucleic acid and its orthologs,alleles, mutants, polymorphic variants, and conservatively modifiedvariants combines the use of synthetic oligonucleotide primers andamplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Inniset al., eds, 1990)). Methods such as polymerase chain reaction (PCR) andligase chain reaction (LCR) can be used to amplify nucleic acidsequences of human T1R directly from mRNA, from cDNA, from genomiclibraries or cDNA libraries. Degenerate oligonucleotides can be designedto amplify T1R homologs using the sequences provided herein. Restrictionendonuclease sites can be incorporated into the primers. Polymerasechain reaction or other in vitro amplification methods may also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of T1R encoding mRNA in physiological samples,for nucleic acid sequencing, or for other purposes. Genes amplified bythe PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

Gene expression of T1R can also be analyzed by techniques known in theart, e.g., reverse transcription and amplification of mRNA, isolation oftotal RNA or poly A⁺ RNA, northern blotting, dot blotting, in situhybridization, RNase protection, high density polynucleotide arraytechnology, e.g., and the like.

Nucleic acids encoding T1R protein can be used with high densityoligonucleotide array technology (e.g., GeneChip™) to identify T1Rprotein, orthologs, alleles, conservatively modified variants, andpolymorphic variants in this invention (see, e.g., Gunthand et al., AIDSRes. Hum. Retroviruses 14: 869-876 (1998); Kozal et al., Nat. Med. 2:753-759 (1996); Matson et al., Anal. Biochem. 224: 110-106 (1995);Lockhart et al., Nat. Biotechnol. 14: 1675-1680 (1996); Gingeras et al.,Genome Res. 8: 435-448 (1998); Hacia et al., Nucleic Acids Res. 26:3865-3866 (1998)).

The gene for T1R is typically cloned into intermediate vectors beforetransformation into prokaryotic or eukaryotic cells for replicationand/or expression. These intermediate vectors are typically prokaryotevectors, e.g., plasmids, or shuttle vectors.

Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene, such as those cDNAsencoding a T1R protein, one typically subclones T1R into an expressionvector that contains a strong promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation. The T1Rnucleic acids can be co-expressed or separately expressed, preferablyco-expressed on the same or a different vector. Suitable bacterialpromoters are well known in the art and described, e.g., in Sambrook etal., and Ausubel et al, supra. Bacterial expression systems forexpressing the T1R protein are available in, e.g., E. coli, Bacillussp., and Salmonella (Palva et al., Gene 22: 229-235 (1983); Mosbach etal., Nature 302: 543-545 (1983). Kits for such expression systems arecommercially available. Eukaryotic expression systems for mammaliancells, yeast, and insect cells are well known in the art and are alsocommercially available. In one preferred embodiment, retroviralexpression systems are used in the present invention.

Selection of the promoter used to direct expression of a heterologousnucleic acid depends on the particular application. The promoter ispreferably positioned about the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the T1R encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding T1R andsignals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. Additional elementsof the cassette may include enhancers and, if genomic DNA is used as thestructural gene, introns with functional splice donor and acceptorsites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as MBP, GST, and LacZ. Epitope tags can also beadded to recombinant proteins to provide convenient methods ofisolation, e.g., c-myc. Sequence tags may be included in an expressioncassette for nucleic acid rescue. Markers such as fluorescent proteins,green or red fluorescent protein, β-gal, CAT, and the like can beincluded in the vectors as markers for vector transduction.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, retroviral vectors, and vectorsderived from Epstein-Barr virus. Other exemplary eukaryotic vectorsinclude pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, andany other vector allowing expression of proteins under the direction ofthe CMV promoter, SV40 early promoter, SV40 later promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

Expression of proteins from eukaryotic vectors can be also be regulatedusing inducible promoters. With inducible promoters, expression levelsare tied to the concentration of inducing agents, such as tetracyclineor ecdysone, by the incorporation of response elements for these agentsinto the promoter. Generally, high level expression is obtained frominducible promoters only in the presence of the inducing agent; basalexpression levels are minimal.

In one embodiment, the vectors of the invention have a regulatablepromoter, e.g., tet-regulated systems and the RU-486 system (see, e.g.,Gossen & Bujard, Proc. Nat'l Acad. Sci. USA 89: 5547 (1992); Oligino etal., Gene Ther. 5: 491-496 (1998); Wang et al., Gene Ther. 4: 432-441(1997); Neering et al., Blood 88: 1147-1155 (1996); and Rendahl et al.,Nat. Biotechnol. 16: 757-761 (1998)). These impart small moleculecontrol on the expression of the candidate target nucleic acids. Thisbeneficial feature can be used to determine that a desired phenotype iscaused by a transfected cDNA rather than a somatic mutation.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase and dihydrofolate reductase. Alternatively,high yield expression systems not involving gene amplification are alsosuitable, such as using a baculovirus vector in insect cells, with a T1Rencoding sequence under the direction of the polyhedrin promoter orother strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of T1R protein,which are then purified using standard techniques (see, e.g., Colley etal., J. Biol. Chem. 264: 17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,biolistics, liposomes, microinjection, plasma vectors, viral vectors andany of the other well known methods for introducing cloned genomic DNA,cDNA, synthetic DNA or other foreign genetic material into a host cell(see, e.g., Sambrook et al., supra). It is only necessary that theparticular genetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingT1R.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofT1R, which is recovered from the culture using standard techniquesidentified below.

Purification of T1R Polypeptides

Either naturally occurring or recombinant T1R polypeptides orT1R3-comprising receptors can be purified for use in functional assays.Naturally occurring T1R proteins or T1R3-comprising receptors can bepurified, e.g,, from human tissue. Recombinant T1R proteins or TR3-comprising receptors can be purified from any suitable expressionsystem. T1R polypeptides are typically co-expressed in the same cell toform T1R3-comprising receptors.

The T1R protein or T1R3-comprising receptor may be purified tosubstantial purity by standard techniques, including selectiveprecipitation with such substances as ammonium sulfate; columnchromatography, immunopurification methods, and others (see, e.g.,Scopes, Protein Purification: Principles and Practice (1982); U.S. Pat.No. 4,673,641; Ausubel et al., supra; and Sambrook et al., supra).

A number of procedures can be employed when recombinant T1R protein orT1R3-comprising receptor is being purified. For example, proteins havingestablished molecular adhesion properties can be reversible fused to theT1R protein or T1R3-comprising receptor. With the appropriate ligand,T1R protein or T1R3-comprising receptor can be selectively adsorbed to apurification column and then freed from the column in a relatively pureform. The fused protein is then removed by enzymatic activity. Finally,T1R protein or T1R3-comprising receptor could be purified usingimmunoaffinity columns.

A. Purification of T1R from Recombinant Bacteria

Recombinant proteins are expressed by transformed bacteria in largeamounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of T1R proteinor T1R3-comprising receptor inclusion bodies. For example, purificationof inclusion bodies typically involves the extraction, separation and/orpurification of inclusion bodies by disruption of bacterial cells, e.g.,by incubation in a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mMMgCl₂, 1 mM DTT, 0.1 mM ATP, and 1 mM PMSF. The cell suspension can belysed using 2-3 passages through a French Press, homogenized using aPolytron (Brinkman Instruments) or sonicated on ice. Alternate methodsof lysing bacteria are apparent to those of skill in the art (see, e.g.,Sambrook et al., supra; Ausubel et al., supra).

If necessary, the inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged to remove unwanted insoluble matter.Proteins that formed the inclusion bodies may be renatured by dilutionor dialysis with a compatible buffer. Suitable solvents include, but arenot limited to urea (from about 4 M to about 8 M), formamide (at leastabout 80%, volume/volume basis), and guanidine hydrochloride (from about4 M to about 8 M). Some solvents which are capable of solubilizingaggregate-forming proteins, for example SDS (sodium dodecyl sulfate),70% formic acid, are inappropriate for use in this procedure due to thepossibility of irreversible denaturation of the proteins, accompanied bya lack of immunogenicity and/or activity. Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing re-formation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art. Human T1R proteins orT1R3-comprising receptors are separated from other bacterial proteins bystandard separation techniques, e.g., with Ni—NTA agarose resin.

Alternatively, it is possible to purify T1R protein or T1R3-comprisingreceptor from bacteria periplasm. After lysis of the bacteria, when theT1R protein or T1R3-comprising receptor is exported into the periplasmof the bacteria, the periplasmic fraction of the bacteria can beisolated by cold osmotic shock in addition to other methods known toskill in the art. To isolate recombinant proteins from the periplasm,the bacterial cells are centrifuged to form a pellet. The pellet isresuspended in a buffer containing 20% sucrose. To lyse the cells, thebacteria are centrifuged and the pellet is resuspended in ice-cold 5 mMMgSO₄ and kept in an ice bath for approximately 10 minutes. The cellsuspension is centrifuged and the supernatant decanted and saved. Therecombinant proteins present in the supernatant can be separated fromthe host proteins by standard separation techniques well known to thoseof skill in the art.

B. Standard Protein Separation Techniques for Purifying T1R ProteinsSolubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of the T1R proteins or T1R3-comprising receptorscan be used to isolate it from proteins of greater and lesser size usingultrafiltration through membranes of different pore size (for example,Amicon or Millipore membranes). As a first step, the protein mixture isultrafiltered through a membrane with a pore size that has a lowermolecular weight cut-off than the molecular weight of the protein ofinterest. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the protein of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed as described below.

Column Chromatography

The T1R proteins or T1R3-comprising receptors can also be separated fromother proteins on the basis of its size, net surface charge,hydrophobicity, and affinity for ligands. In addition, antibodies raisedagainst proteins can be conjugated to column matrices and the proteinsimmunopurified. All of these methods are well known in the art. It willbe apparent to one of skill that chromatographic techniques can beperformed at any scale and using equipment from many differentmanufacturers (e.g., Pharmacia Biotech).

Assays for Modulators of T1R Protein

A. Assays

Modulation of a T1R3-comprising sweet taste receptor, and correspondingmodulation of taste, can be assessed using a variety of in vitro and invivo assays. Such assays can be used to test for inhibitors andactivators of T1R3-comprising sweet taste receptors, and, consequently,inhibitors and activators of taste. Such modulators of T1R3-comprisingsweet taste receptors, which are involved in taste signal transduction.Modulators of T1R3-comprising sweet taste receptors are tested usingeither recombinant or naturally occurring T1R3-comprising sweet tastereceptors, preferably human receptors.

Preferably, the T1R3-comprising sweet taste receptor will have asequence as encoded by a sequence provided herein or a conservativelymodified variant thereof. Alternatively, the T1R3-comprising sweet tastereceptor of the assay will be derived from a eukaryote and include anamino acid subsequence having substantial amino acid sequence identityto the sequences provided herein or is encoded by a nucleotide sequencethat hybridizes under stringent conditions (moderate or high) to anucleotide sequence as described herein. Generally, the amino acidsequence identity will be at least 60%, preferably at least 65%, 70%,75%, 80%, 85%, or 90%, most preferably at least 95%.

Measurement of sweet taste signal transduction or loss-of-sweet tastesignal transduction phenotype on T1R3-comprising sweet taste receptor orcell expressing the T1R3-comprising sweet taste receptor, eitherrecombinant or naturally occurring, can be performed using a variety ofassays, in vitro, in vivo, and ex vivo, as described herein. A suitablephysical, chemical or phenotypic change that affects activity or bindingcan be used to assess the influence of a test compound on thepolypeptide of this invention. When the functional effects aredetermined using intact cells or animals, one can also measure a varietyof effects such as, in the case of signal transduction, e.g., ligandbinding, hormone release, transcriptional changes to both known anduncharacterized genetic markers (e.g., northern blots), changes in cellmetabolism such as pH changes, and changes in intracellular secondmessengers such as Ca²⁺, IP3, cGMP, or cAMP.

In Vitro Assays

Assays to identify compounds with T1R3-comprising sweet taste receptormodulating activity can be performed in vitro. Such assays can use afull length T1R3-comprising sweet taste receptor or a variant thereof,or a fragment of a T1R3-comprising sweet taste receptor, such as anextracellular domain, fused to a heterologous protein to form a chimera.Purified recombinant or naturally occurring T1R3-comprising sweet tastereceptor can be used in the in vitro methods of the invention. Inaddition to purified T1R3-comprising sweet taste receptor, therecombinant or naturally occurring T1R3-comprising sweet taste receptorcan be part of a cellular lysate or a cell membrane. As described below,the binding assay can be either solid state or soluble. Preferably, theprotein or membrane is bound to a solid support, either covalently ornon-covalently. Often, the in vitro assays of the invention are ligandbinding or ligand affinity assays, either non-competitive or competitive(with known extracellular ligands as described herein, or with a knownintracellular ligand GTP). Other in vitro assays include measuringchanges in spectroscopic (e g., fluorescence, absorbance, refractiveindex), hydrodynamic (e.g., shape), chromatographic, or solubilityproperties for the protein.

In one embodiment, a high throughput binding assay is performed in whichthe T1R3-comprising sweet taste receptor or chimera comprising afragment thereof is contacted with a potential modulator and incubatedfor a suitable amount of time. In one embodiment, the potentialmodulator is bound to a solid support, and the T1R3-comprising sweettaste receptor is added. In another embodiment, the T1R3-comprisingsweet taste receptor is bound to a solid support. A wide variety ofmodulators can be used, as described below, including small organicmolecules, peptides, antibodies, and T1R3-comprising sweet tastereceptor ligand analogs. A wide variety of assays can be used toidentify T1R3-comprising sweet taste receptor-modulator binding,including labeled protein-protein binding assays, electrophoreticmobility shifts, immunoassays, enzymatic assays such as phosphorylationassays, and the like. In some cases, the binding of the candidatemodulator is determined through the use of competitive binding assays,where interference with binding of a known ligand is measured in thepresence of a potential modulator. Ligands for T1R3-comprising sweettaste receptors are provided herein. Either the modulator or the knownligand is bound first, and then the competitor is added. After theT1R3-comprising sweet taste receptor is washed, interference withbinding, either of the potential modulator or of the known ligand, isdetermined. Often, either the potential modulator or the known ligand islabeled.

Cell-Based In Vivo Assays

In another embodiment, a T1R3-comprising sweet taste receptor isexpressed in a cell (e.g., by co-expression two heterologous members ofthe T1R family such as T1R1 and T1R3 or T1R2 and T1R3), and functional,e.g., physical and chemical or phenotypic, changes are assayed toidentify T1R3-comprising sweet taste receptor taste modulators. Cellsexpressing T1R3-comprising sweet taste receptor can also be used inbinding assays. Any suitable functional effect can be measured, asdescribed herein. For example, ligand binding, G-protein binding, andGPCR signal transduction, e.g., changes in intracellular Ca²⁺ levels,are all suitable assays to identify potential modulators using a cellbased system. Suitable cells for such cell based assays include bothprimary cells and cell lines, as described herein. The T1R3-comprisingsweet taste receptor can be naturally occurring or recombinant. Also, asdescribed above, chimeric T1R3-comprising sweet taste receptors withGPCR activity can be used in cell based assays. For example, theextracellular domain of an T1R protein can be fused to the transmembraneand/or cytoplasmic domain of a heterologous protein, preferably aheterologous GPCR. Such a chimeric GPCR would have GPCR activity andcould be used in cell based assays of the invention.

In another embodiment, cellular T1R polypeptide levels are determined bymeasuring the level of protein or mRNA. The level of T1R protein orproteins related to T1R signal transduction are measured usingimmunoassays such as western blotting, ELISA and the like with anantibody that selectively binds to the T1R3-compri sing sweet tastereceptor or a fragment thereof. For measurement of mRNA, amplification,e.g., using PCR, LCR, or hybridization assays, e.g., northernhybridization, RNAse protection, dot blotting, are preferred. The levelof protein or mRNA is detected using directly or indirectly labeleddetection agents, e.g., fluorescently or radioactively labeled nucleicacids, radioactively or enzymatically labeled antibodies, and the like,as described herein.

Alternatively, T1R3-comprising receptor expression can be measured usinga reporter gene system. Such a system can be devised using an T1Rprotein promoter operably linked to a reporter gene such aschloramphenicol acetyltransferase, firefly luciferase, bacterialluciferase, β-galactosidase and alkaline phosphatase. Furthermore, theprotein of interest can be used as an indirect reporter via attachmentto a second reporter such as red or green fluorescent protein (see,e.g., Mistili & Spector, Nature Biotechnology 15: 961-964 (1997)). Thereporter construct is typically transfected into a cell. After treatmentwith a potential modulator, the amount of reporter gene transcription,translation, or activity is measured according to standard techniquesknown to those of skill in the art.

In another embodiment, a functional effect related to GPCR signaltransduction can be measured. An activated or inhibited T1R3-comprisingG-coupled protein receptor will alter the properties of target enzymes,second messengers, channels, and other effector proteins. The examplesinclude the activation of cGMP phosphodiesterase, adenylate cyclase,phospholipase C, IP3, and modulation of diverse channels by G proteins.Downstream consequences can also be examined such as generation ofdiacyl glycerol and IP3 by phospholipase C, and in turn, for calciummobilization by IP3. Activated GPCR receptors become substrates forkinases that phosphorylate the C-terminal tail of the receptor (andpossibly other sites as well). Thus, activators will promote thetransfer of ³²P from gamma-labeled GTP to the receptor, which can beassayed with a scintillation counter. The phosphorylation of theC-terminal tail will promote the binding of arrestin-like proteins andwill interfere with the binding of G-proteins. For a general review ofGPCR signal transduction and methods of assaying signal transduction,see, e.g., Methods in Enzymology, vols. 237 and 238 (1994) and volume 96(1983); Bourne et al., Nature 10: 349: 117-27 (1991); Bourne et al.,Nature 348: 125-32 (1990); Pitcher et al., Annu. Rev. Biochem. 67:653-92 (1998).

As described above, activation of some G-protein coupled receptorsstimulates the formation of inositol triphosphate (IP3) throughphospholipase C-mediated hydrolysis of phosphatidylinositol (Berridge &Irvine, Nature 312: 315-21 (1984)). IP3 in turn stimulates the releaseof intracellular calcium ion stores. Thus, a change in cytoplasmiccalcium ion levels, or a change in second messenger levels such as IP3can be used to assess G-protein coupled receptor function. Cellsexpressing such G-protein coupled receptors may exhibit increasedcytoplasmic calcium levels as a result of contribution from bothintracellular stores and via activation of ion channels, in which caseit may be desirable although not necessary to conduct such assays incalcium-free buffer, optionally supplemented with a chelating agent suchas EGTA, to distinguish fluorescence response resulting from calciumrelease from internal stores.

In one example, T1R3-comprising sweet taste receptor GPCR activity ismeasured by expressing a T1R3-comprising sweet taste receptor in aheterologous cell with a promiscuous G-protein that links the receptorto a phospholipase C signal transduction pathway (see Offermanns &Simon, J. Biol. Chem. 270: 15175-15180 (1995)). Modulation of signaltransduction is assayed by measuring changes in intracellular Ca²⁺levels, which change in response to modulation of the GPCR signaltransduction pathway via administration of a molecule that associateswith an T1R3-comprising sweet taste receptor.

Changes in Ca²⁺ levels are optionally measured using fluorescent Ca²⁺indicator dyes and fluorometric imaging.

In another example, phosphatidyl inositol (PI) hydrolysis can beanalyzed according to U.S. Pat. No. 5,436,128, herein incorporated byreference. Briefly, the assay involves labeling of cells with³H-myoinositol for 48 or more hrs. The labeled cells are treated with atest compound for one hour. The treated cells are lysed and extracted inchloroform-methanol-water after which the inositol phosphates wereseparated by ion exchange chromatography and quantified by scintillationcounting. Fold stimulation is determined by calculating the ratio of cpmin the presence of agonist to cpm in the presence of buffer control.Likewise, fold inhibition is determined by calculating the ratio of cpmin the presence of antagonist to cpm in the presence of buffer control(which may or may not contain an agonist).

Other assays can involve determining the activity of receptors which,when activated, result in a change in the level of intracellular cyclicnucleotides, e.g., cAMP or cGMP, by activating or inhibiting enzymessuch as adenylate cyclase. In cases where activation of the receptorresults in a decrease in cyclic nucleotide levels, it may be preferableto expose the cells to agents that increase intracellular cyclicnucleotide levels, e.g., forskolin, prior to adding areceptor-activating compound to the cells in the assay.

In one example, the changes in intracellular cAMP or cGMP can bemeasured using immunoassays. The method described in Offermanns & Simon,J. Biol. Chem. 270: 15175-15180 (1995) may be used to determine thelevel of cAMP. Also, the method described in Felley-Bosco et al., Am. J.Resp. Cell and Mol. Biol. 11: 159-164 (1994) may be used to determinethe level of cGMP. Further, an assay kit for measuring cAMP and/or cGMPis described in U.S. Pat. No. 4,115,538, herein incorporated byreference.

In one example, assays for G-protein coupled receptor activity includecells that are loaded with ion or voltage sensitive dyes to reportreceptor activity. Assays for determining activity of such receptors canalso use known agonists and antagonists for other 30 G-protein coupledreceptors as negative or positive controls to assess activity of testedcompounds. In assays for identifying modulatory compounds (e.g.,agonists, antagonists), changes in the level of ions in the cytoplasm ormembrane voltage will be monitored using an ion sensitive or membranevoltage fluorescent indicator, respectively. Among the ion-sensitiveindicators and voltage probes that may be employed are those disclosedin the Molecular Probes 1997 Catalog. For G-protein coupled receptors,promiscuous G-proteins such as Gα15 and Gα16 can be used in the assay ofchoice (Wilkie et al., Proc. Nat'l Acad. Sci. USA 88: 10049-10053(1991)). Such promiscuous G-proteins allow coupling of a wide range ofreceptors.

Animal Models

Animal models of taste also find use in screening for modulators oftaste, such as the Sac taster and non-taster mouse strains as describedherein. Similarly, transgenic animal technology including gene knockouttechnology, for example as a result of homologous recombination with anappropriate gene targeting vector, or gene overexpression, will resultin the absence or increased expression of the T1R3-comprising receptoror components thereof. When desired, tissue-specific expression orknockout of the T1R3-comprising receptors or components thereof may benecessary. Transgenic animals generated by such methods find use asanimal models of taste modulation and are additionally useful inscreening for modulators of taste modulation.

B. Modulators

The compounds tested as modulators of T1R3-comprising sweet tastereceptors can be any small organic molecule, or a biological entity,such as a protein, e.g., an antibody or peptide, a sugar, a nucleicacid, e.g., an antisense oligonucleotide or a ribozyme, or a lipid.Alternatively, modulators can be genetically altered versions of aT1R3-comprising sweet taste receptor. Typically, test compounds will besmall organic molecules, peptides, lipids, and lipid analogs.

Essentially any chemical compound can be used as a potential modulatoror ligand in the assays of the invention, although most often compoundscan be dissolved in aqueous or organic (especially DMSO-based) solutionsare used. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial small organic molecule or peptide librarycontaining a large number of potential therapeutic compounds (potentialmodulator or ligand compounds). Such “combinatorial chemical libraries”or “ligand libraries” are then screened in one or more assays, asdescribed herein, to identify those library members (particular chemicalspecies or subclasses) that display a desired characteristic activity.The compounds thus identified can serve as conventional “lead compounds”or can themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37: 487-493(1991) and Houghton et al., Nature 354: 84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90: 6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114: 6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114: 9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116: 2661 (1994)),oligocarbamates (Cho et al., Science 261: 1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59: 658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g.,Liang et al., Science, 274: 1520-1522 (1996) and U.S. Pat. No.5,593,853), small organic molecule libraries (see, e.g.,benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids,U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat.No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S.Pat. No. 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy.; Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md., etc.).

C. Solid State and Soluble High Throughput Assays

In one embodiment the invention provides soluble assays using aT1R3-comprising sweet taste receptor, or a cell or tissue expressing aT1R3-comprising sweet taste receptor, either naturally occurring orrecombinant. In another embodiment, the invention provides solid phasebased in vitro assays in a high throughput format, where theT1R3-comprising sweet taste receptor is attached to a solid phasesubstrate. Any one of the assays described herein can be adapted forhigh throughput screening, e.g., ligand binding, cellular proliferation,cell surface marker flux, e.g., screening, radiolabeled GTP binding,second messenger flux, e.g., Ca²⁺, IP3, cGMP, or cAMP, cytokineproduction, etc.

In the high throughput assays of the invention, either soluble or solidstate, it is possible to screen up to several thousand differentmodulators or ligands in a single day. This methodology can be used forT1R3-comprising sweet taste receptors in vitro, or for cell-based ormembrane-based assays comprising T1R3-comprising sweet taste receptors.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100- about 1500different compounds. It is possible to assay many plates per day; assayscreens for up to about 6,000, 20,000, 50,000, or more than 100,000different compounds are possible using the integrated systems of theinvention.

For a solid state reaction, the protein of interest or a fragmentthereof, e.g., an extracellular domain, or a cell or membrane comprisingthe protein of interest or a fragment thereof as part of a fusionprotein can be bound to the solid state component, directly orindirectly, via covalent or non covalent linkage e.g., via a tag. Thetag can be any of a variety of components. In general, a molecule whichbinds the tag (a tag binder) is fixed to a solid support, and the taggedmolecule of interest is attached to the solid support by interaction ofthe tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.)Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders; see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto persons of skill in the art. For example, poly(ethelyne glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc. 85: 2149-2154 (1963) (describing solid phase synthesisof, e.g., peptides); Geysen et al., J. Immun. Meth. 102: 259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron 44: 60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4): 718-719(1993); and Kozal et al., Nature Medicine 2(7): 753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

Immunological Detection of T1R3-Comprising Receptors

In addition to the detection of T1R genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect T1R3-comprising sweet taste receptors of the invention. Suchassays are useful for screening for modulators of T1R3-comprising sweettaste receptors, as well as for therapeutic and diagnostic applications.Immunoassays can be used to qualitatively or quantitatively analyzeT1R3-comprising sweet taste receptors. A general overview of theapplicable technology can be found in Harlow & Lane, Antibodies: ALaboratory Manual (1988).

A. Production of Antibodies

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with the T1R proteins and T1R3-comprising sweet tastereceptors are known to those of skill in the art (see, e.g., Coligan,Current Protocols in Immunology (1991); Harlow & Lane, supra; Goding,Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler& Milstein, Nature 256: 495-497 (1975). Such techniques include antibodypreparation by selection of antibodies from libraries of recombinantantibodies in phage or similar vectors, as well as preparation ofpolyclonal and monoclonal antibodies by immunizing rabbits or mice (see,e.g., Huse et al., Science 246: 1275-1281 (1989); Ward et al., Nature341: 544-546 (1989)).

A number of immunogens comprising portions of T1R protein orT1R3-comprising sweet taste receptor may be used to produce antibodiesspecifically reactive with T1R protein. For example, recombinant T1Rprotein or an antigenic fragment thereof, can be isolated as describedherein. Recombinant protein can be expressed in eukaryotic orprokaryotic cells as described above, and purified as generallydescribed above. Recombinant protein is the preferred immunogen for theproduction of monoclonal or polyclonal antibodies. Alternatively, asynthetic peptide derived from the sequences disclosed herein andconjugated to a carrier protein can be used an immunogen. Naturallyoccurring protein may also be used either in pure or impure form. Theproduct is then injected into an animal capable of producing antibodies.Either monoclonal or polyclonal antibodies may be generated, forsubsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to the beta subunits.When appropriately high titers of antibody to the immunogen areobtained, blood is collected from the animal and antisera are prepared.Further fractionation of the antisera to enrich for antibodies reactiveto the protein can be done if desired (see, Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see, Kohler & Milstein, Eur. J. Immunol. 6: 511-519(1976)). Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodswell known in the art. Colonies arising from single immortalized cellsare screened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse, etal., Science 246: 1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-T1R orT1R3-comprising sweet taste receptor proteins, using a competitivebinding immunoassay. Specific polyclonal antisera and monoclonalantibodies will usually bind with a K_(d) of at least about 0.1 mM, moreusually at least about 1 μM, preferably at least about 0.1 μM or better,and most preferably, 0.01 μM or better. Antibodies specific only for aparticular T1R3-comprising sweet taste receptor ortholog, such as humanT1R3-comprising sweet taste receptor, can also be made, by subtractingout other cross-reacting orthologs from a species such as a non-humanmammal. In addition, individual T1R proteins can be used to subtract outantibodies that bind both to the receptor and the individual T1Rproteins. In this manner, antibodies that bind only to a heterodimericreceptor may be obtained.

Once the specific antibodies against T1R3-comprising sweet tastereceptors are available, the protein can be detected by a variety ofimmunoassay methods. In addition, the antibody can be usedtherapeutically as a T1R3-comprising sweet taste receptor modulators.For a review of immunological and immunoassay procedures, see Basic andClinical Immunology (Stites & Terr eds., 7^(th) ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio, ed., 1980); and Harlow & Lane, supra.

B. Immunological Binding Assays

T1R3-comprising sweet taste receptors can be detected and/or quantifiedusing any of a number of well recognized immunological binding assays(see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and4,837,168). For a review of the general immunoassays, see also Methodsin Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993);Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991).Immunological binding assays (or immunoassays) typically use an antibodythat specifically binds to a protein or antigen of choice (in this casethe T1R3-comprising sweet taste receptor or antigenic subsequencethereof). The antibody (e.g., anti-T1R3-comprising sweet taste receptor)may be produced by any of a number of means well known to those of skillin the art and as described above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled T1R3-comprising sweettaste receptor or a labeled anti-T1R3-comprising sweet taste receptorantibody. Alternatively, the labeling agent may be a third moiety, sucha secondary antibody, that specifically binds to the antibody/T1R3-comprising sweet taste receptor complex (a secondary antibody istypically specific to antibodies of the species from which the firstantibody is derived). Other proteins capable of specifically bindingimmunoglobulin constant regions, such as protein A or protein G may alsobe used as the label agent. These proteins exhibit a strongnon-immunogenic reactivity with immunoglobulin constant regions from avariety of species (see, e.g., Kronval et al., J. Immunol. 111:1401-1406 (1973); Akerstrom et al., J. Immunol. 135: 2589-2542 (1985)).The labeling agent can be modified with a detectable moiety, such asbiotin, to which another molecule can specifically bind, such asstreptavidin. A variety of detectable moieties are well known to thoseskilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, optionally from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Non-Competitive Assay Formats

Immunoassays for detecting T1R3-comprising sweet taste receptors insamples may be either competitive or noncompetitive. Noncompetitiveimmunoassays are assays in which the amount of antigen is directlymeasured. In one preferred “sandwich” assay, for example, theanti-T1R3-comprising sweet taste receptor antibodies can be bounddirectly to a solid substrate on which they are immobilized. Theseimmobilized antibodies then capture T1R3-comprising sweet tastereceptors present in the test sample. T1R3-comprising sweet tastereceptors thus immobilized are then bound by a labeling agent, such as asecond T1R3-comprising sweet taste receptor antibody bearing a label.Alternatively, the second antibody may lack a label, but it may, inturn, be bound by a labeled third antibody specific to antibodies of thespecies from which the second antibody is derived. The second or thirdantibody is typically modified with a detectable moiety, such as biotin,to which another molecule specifically binds, e.g., streptavidin, toprovide a detectable moiety.

Competitive Assay Formats

In competitive assays, the amount of T1R3-comprising sweet tastereceptor present in the sample is measured indirectly by measuring theamount of a known, added (exogenous) T1R3-comprising sweet tastereceptor displaced (competed away) from an anti-T1R3-comprising sweettaste receptor antibody by the unknown T1R3-comprising sweet tastereceptor present in a sample. In one competitive assay, a known amountof T1R3-comprising sweet taste receptor is added to a sample and thesample is then contacted with an antibody that specifically binds to aT1R3-comprising sweet taste receptor. The amount of exogenousT1R3-comprising sweet taste receptor bound to the antibody is inverselyproportional to the concentration of T1R3-comprising sweet tastereceptor present in the sample. In a particularly preferred embodiment,the antibody is immobilized on a solid substrate. The amount ofT1R3-comprising sweet taste receptor bound to the antibody may bedetermined either by measuring the amount of T1R3-comprising sweet tastereceptor present in a T1R3-comprising sweet taste receptor/antibodycomplex, or alternatively by measuring the amount of remaininguncomplexed protein. The amount of T1R3-comprising sweet taste receptormay be detected by providing a labeled T1R3-comprising sweet tastereceptor molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known T1R3-comprising sweet taste receptor is immobilizedon a solid substrate. A known amount of anti-T1R3-comprising sweet tastereceptor antibody is added to the sample, and the sample is thencontacted with the immobilized T1R3-comprising sweet taste receptor. Theamount of anti- T1R3-comprising sweet taste receptor antibody bound tothe known immobilized T1R3-comprising sweet taste receptor is inverselyproportional to the amount of T1R3-comprising sweet taste receptorpresent in the sample. Again, the amount of immobilized antibody may bedetected by detecting either the immobilized fraction of antibody or thefraction of the antibody that remains in solution. Detection may bedirect where the antibody is labeled or indirect by the subsequentaddition of a labeled moiety that specifically binds to the antibody asdescribed above.

Cross-Reactivity Determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations. For example, a T1R3-comprising sweettaste receptor can be immobilized to a solid support. Proteins (e.g.,T1R3-comprising sweet taste receptors and homologs) are added to theassay that compete for binding of the antisera to the immobilizedantigen. The ability of the added proteins to compete for binding of theantisera to the immobilized protein is compared to the ability of theT1R3-comprising sweet taste receptor to compete with itself. The percentcrossreactivity for the above proteins is calculated, using standardcalculations. Those antisera with less than 10% crossreactivity witheach of the added proteins listed above are selected and pooled. Thecross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added considered proteins, e.g.,distantly related homologs.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps an allele or polymorphic variant of aT1R3-comprising sweet taste receptor, to the immunogen protein. In orderto make this comparison, the two proteins are each assayed at a widerange of concentrations and the amount of each protein required toinhibit 50% of the binding of the antisera to the immobilized protein isdetermined. If the amount of the second protein required to inhibit 50%of binding is less than 10 times the amount of the T1R3-comprising sweettaste receptor that is required to inhibit 50% of binding, then thesecond protein is said to specifically bind to the polyclonal antibodiesgenerated to a T1R3-comprising sweet taste receptor immunogen.

Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of T1R3-comprising sweet taste receptors in the sample. Thetechnique generally comprises separating sample proteins by gelelectrophoresis on the basis of molecular weight, transferring theseparated proteins to a suitable solid support, (such as anitrocellulose filter, a nylon filter, or derivatized nylon filter), andincubating the sample with the antibodies that specifically bindT1R3-comprising sweet taste receptors. The anti-T1R3-comprising sweettaste receptor antibodies specifically bind to the T1R3-comprising sweettaste receptor on the solid support. These antibodies may be directlylabeled or alternatively may be subsequently detected using labeledantibodies (e.g., labeled sheep anti-mouse antibodies) that specificallybind to the anti-T1R3-comprising sweet taste receptor antibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5: 34-41 (1986)).

Reduction of Non-Specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and calorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize T1R3-comprisingsweet taste receptors, or secondary antibodies that recognizeanti-T1R3-comprising sweet taste receptor.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

Pharmaceutical Compositions and Administration

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered (e.g., nucleic acid,oligonucleotide, protein, peptide, small organic molecule, lipid,carbohydrate, particle, or transduced cell), as well as by theparticular method used to administer the composition. Accordingly, thereare a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention (see, e.g., Remington'sPharmaceutical Sciences, 17^(th) ed., 1989). Administration can be inany convenient manner, e.g., by injection, oral administration,inhalation, transdermal application, or rectal administration.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, microcrystalline cellulose,gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearicacid, and other excipients, colorants, fillers, binders, diluents,buffering agents, moistening agents, preservatives, flavoring agents,dyes, disintegrating agents, and pharmaceutically compatible carriers.Lozenge forms can comprise the active ingredient in a flavor, e.g.,sucrose, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

The compound of choice, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of commends can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by nucleic acids for ex vivo therapy can also be administeredintravenously or parenterally as described above.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, or transduced cell type in aparticular patient.

In determining the effective amount of the vector to be administered inthe treatment or prophylaxis of conditions owing to diminished oraberrant expression of a T1R3-comprising sweet taste receptor, thephysician evaluates circulating plasma levels of the vector, vectortoxicities, progression of the disease, and the production ofanti-vector antibodies. In general, the dose equivalent of a nakednucleic acid from a vector is from about 1 μg to 100 μg for a typical 70kilogram patient, and doses of vectors which include a retroviralparticle are calculated to yield an equivalent amount of therapeuticnucleic acid.

For administration, compounds and transduced cells of the presentinvention can be administered at a rate determined by the LD-50 of theinhibitor, vector, or transduced cell type, and the side-effects of theinhibitor, vector or cell type at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

Cellular Transfection and Gene Therapy

The present invention provides the nucleic acids of T1R3-comprisingsweet taste receptors for the transfection of cells in vitro and invivo. These nucleic acids can be inserted into any of a number ofwell-known vectors for the transfection of target cells and organisms asdescribed below. The nucleic acids are transfected into cells, ex vivoor in vivo, through the interaction of the vector and the target cell.The nucleic acid, under the control of a promoter, then expresses aT1R3-comprising sweet taste receptor of the present invention, byco-expressing two members of the T1R family, thereby mitigating theeffects of absent, partial inactivation, or abnormal expression of aT1R3-comprising sweet taste receptor. The compositions are administeredto a patient in an amount sufficient to elicit a therapeutic response inthe patient. An amount adequate to accomplish this is defined as“therapeutically effective dose or amount.”

Such gene therapy procedures have been used to correct acquired andinherited genetic defects and other diseases in a number of contexts.The ability to express artificial genes in humans facilitates theprevention and/or cure of many important human diseases, including manydiseases which are not amenable to treatment by other therapies (for areview of gene therapy procedures, see Anderson, Science 256: 808-813(1992); Nabel & Felgner, TIBTECH 11: 211-217 (1993); Mitani & Caskey,TIBTECH 11: 162-166 (1993); Mulligan, Science 926-932 (1993); Dillon,TIBTECH 11: 167-175 (1993); Miller, Nature 357: 455-460 (1992); VanBrunt, Biotechnology 6(10): 1149-1154 (1998); Vigne, RestorativeNeurology and Neuroscience 8: 35-36 (1995); Kremer & Perricaudet,British Medical Bulletin 51(1): 31-44 (1995); Haddada et al., in CurrentTopics in Microbiology and Immunology (Doerfler & Bohm eds., 1995); andYu et al., Gene Therapy 1: 13-26 (1994)).

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EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Results

T1R3 is Encoded by the Sac Locus

In previous studies, we identified two novel G protein-coupled receptorsof the T1R family, T1R1 and T1R2, that are selectively expressed insubsets of taste receptor cells of the tongue and palate epithelium(Hoon et al., 1999; Genbank Accession numbers: AY032620-AY032623). Wealso previously identified functional bitter taste receptor genes, theT2R family (Adler et al., 2000; Chandrashekar et al., 2000). Both T1R1and T1R2 were initially mapped to the distal end of chromosome 4, in theproximity of Sac (Hoon et al., 1999). However, radiation hybrid analysisand high-resolution genetic mapping separated these receptors from theSac genetic interval (Li et al., 2001), thus eliminating them ascandidate Sac genes (FIG. 1). Recently, six independent groups reportedthat a related receptor gene, T1R3, is tightly linked to the Sac locus(Kitagawa et al., 2001; Max et al., 2001; Montmayeur et al., 2001; Sainzet al., 2001 and press releases from Senomyx, La Jolla, Calif. and Li etal., 2001 Achems XXIII, Sarasota Fla.), and that polymorphic variants ofT1R3 co-segregate with Sac taster and non-taster alleles. This geneticlinkage was used to hypothesize that T1R3 corresponds to the Sac gene.We also isolated and characterized T1R3, and reasoned that if Sac infact encodes T1R3, then introduction of a taster allele of thiscandidate receptor should rescue the taste deficit of Sac non-tastermice.

A 15 kb genomic clone containing the T1R3 sequence from a Sac tasterstrain (C57 BL/6) was used to engineer a transgenic rescue construct(FIG. 2 a). In order to follow the presence and expression of thetransgene versus the endogenous T1R3 allele, we replaced its 3′-UTR andpoly-adenylation signal with that of bovine growth hormone. Our strategywas to produce progeny that were homozygous for the T1R3 non-tasterallele, but carried the taster-derived transgene. We obtained 4 foundermice, and two independent lines were examined for appropriate expressionof the transgene and assayed for behavioral rescue of sucrose andsaccharin tasting (Fuller, 1974). Age- and sex-matched siblings thatlacked the transgene were used as controls in all experiments. FIG. 2 billustrates that all the cells expressing the endogenous T1R3 receptor,and only these cells, also express the transgene (identical results wereobtained in taster and non-taster genetic backgrounds; data not shown).

If the T1R3 taster allele rescues the taste deficiency of Sacnon-tasters, their saccharin and sucrose dose-responses should beshifted to recapitulate the sensitivity seen in Sac taster animals(Fuller, 1974; Bachmanov et al., 1997). FIG. 2 demonstrates that theT1R3 transgene fully rescues the taste defect of Sac non-tasters.Animals without a transgene are indistinguishable from non-taster 129/Svcontrol mice (FIG. 2 c and d, open black circles). In contrast, siblingswith the same Sac non-taster background but expressing the transgene arenow equivalent to taster C57BL/6 control mice (FIG. 2 c and d, redtraces). The presence of the transgene did not influence other tastemodalities (FIG. 2 e-h), nor did it alter the sweet sensitivity oftaster strains (data not shown). Equivalent results were obtained withthe two independent transgenic lines. These results validate T1R3 as theSac locus, and suggest that T1R3 may function as a sweet taste receptor.

Expression of T1Rs

Recently, T1R3 was shown to be expressed in subsets of taste receptorcells in various taste papillae (Kitagawa et al., 2001; Max et al.,2001; Montmayeur et al., 2001; Sainz et al., 2001). However, there weresignificant discrepancies between the reported patterns of expression,with results ranging from little if any expression at the front of thetongue (fungiform papillae; Sainz et al., 2001) to significantexpression in all taste buds (Kitagawa et al., 2001). We examined theexpression of T1R3 in circumvallate, foliate, fungiform and palate tastebuds and find that T1R3 is expressed in ˜30% of cells from all types oftaste buds (FIG. 3; see also Montmayeur et al., 2001). This topographicpattern of expression closely approximates the aggregate of T1R1 andT1R2 expression (FIG. 3, Hoon et al., 1999), and suggests possibleco-expression of T1R1 with T1R3 and T1R2 with T1R3. The co-expression ofT1R2 and T1R3 in circumvallate (Max et al., 2001; Montmayeur et al.,2001) and foliate papillae (Montmayeur et al., 2001) was recentlyexamined by RT-PCR and by in situ hybridization, but a comprehensivestudy of all three T1Rs in the different classes of taste buds waslacking. Thus, we performed double labeling experiments using two-colorfluorescent in situ hybridization. Our results demonstrated that T1R3 isco-expressed with T1R2 in all circumvallate, foliate and palate tastebuds, with every T1R2-positive cell also expressing T1R3 (FIG. 4).Similarly, T1R1 is co-expressed with T1R3 in fungiform and palate tastereceptor cells. However, there is also a fraction of cells withnon-overlapping expression of T1R3 in fungiform and palate taste buds.Therefore, we can define three major classes of cell types based ontheir T1R expression profiles: T1R1 and T1R3 (T1R1+3), T1R2 and T1R3(T1R2+3) and T1R3.

T1Rs Encode Functional Sweet Taste Receptors

Demonstration that T1Rs encode sweet receptors requires functionalvalidation. To monitor translocation of receptors to the plasma membranewe raised antibodies against T1R1, T1R2 and T1R3, and tested expressionof native and epitope-tagged mouse, human and rat receptors in varioustissue culture cell lines. We observed that rat T1Rs were expressedefficiently; we therefore used the rat genes in all heterologousexpression studies. To assay function, we expressed T1Rs with a Gα16-Gzchimera and Gα15, two G-protein α-subunits that together efficientlycouple Gs, Gi, Gq and gustducin-linked receptors to phospholipase Cβ(Offermanns and Simon, 1995; Krautwurst et al., 1998; Chandrashekar etal., 2000; Mody et al., 2000). In this system, receptor activation leadsto increases in intracellular calcium [Ca²⁺]i, which can be monitored atthe single cell level using the FURA-2 calcium-indicator dye (Tsien etal., 1985).

Because of the apparent co-expression of T1R1 or T1R2 with T1R3, wetransfected various rat T1Rs singly and in combinations (forco-expression) into HEK-293 cells expressing the promiscuous G═15 andGα16-Gz proteins. After loading the cells with FURA-2, we assayed forresponses to a wide range of sweet tastants, including sugars, aminoacids, and artificial sweeteners; we also tested several bitter tastants(see Experimental Procedures). Cells expressing rat T1R2 and T1R3(T1R2+3) robustly responded to a subset of sweet compounds includingsucrose, fructose, saccharin (but not to N-methyl-saccharin, a non-sweetsaccharin derivative), acesulfame-K, dulcin, and two novel intenselysweet compounds (Nagarajan et al., 1996, guanidinoacetic acid 1 and 2,referred to as GA-1 and GA-2; FIGS. 5 and 6 a). The responses werereceptor- and Gα-dependent because cells lacking either of thesecomponents did not trigger [Ca²⁺]i changes, even at vastly higherconcentrations of tastants (FIG. 5). Notably, the activation of T1R2+3is extremely selective. On the one hand, this receptor combination didnot respond to a large number of mono- and disaccharides and artificialsweeteners, including glucose, galactose, maltose and aspartame (FIG. 6a). On the other hand, the response was dependent on the presence ofboth T1R2 and T1R3; either receptor alone did not respond to any of thecompounds assayed in these studies, even at concentrations that farexceeded their biologically relevant range of action (data not shown).These results demonstrate that T1R2 and T1R3, when co-expressed in thesame cell, function as a sweet taste receptor.

Evidence that association of the polypeptides, or heteromerization, isrequired for the formation of a functional T1R receptor was obtained byco-expression of a dominant negative T1R. Co-transfection of wild typeT1R2 and T1R3 with a T1R2 receptor harboring a C-terminal truncation(Salahpour et al., 2000) nearly abolished the T1R2+3 responses (>85%reduction, data not shown).

If the responses of T1R2+3 reflect the function of the native sweetreceptor, we reasoned that the sensitivity thresholds seen in thecell-based assays should parallel the behavioral thresholds fordetection of these sweet tastants in vivo. Indeed, FIG. 6 b showsdose-responses for GA-2 (in vivo threshold ˜2 μM), saccharin (in vivothreshold ˜0.5 mM), acesulfame-K (in vivo threshold ˜0.5 mM) and sucrose(in vivo threshold ˜20 mM), demonstrating a good match between thecell-based responses and their biological threshold. No responses weredetected against a panel of bitter tastants, or umami stimuli.

To examine the sweet taste responses in detail, cells transfected withT1R2+3 were placed on a microperfusion chamber and superfused with testsolutions under various conditions. FIG. 6 c shows that responses to thesweet tastants closely follow application of the stimulus (latency<1 s).As expected, when the tastant was removed, [Ca²⁺]i returned to baseline.A prolonged exposure to the sweet compound (>10 s) resulted inadaptation: a fast increase of [Ca²⁺]i followed by a rapid, butincomplete decline to the resting level. Similarly, successiveapplications of the tastant led to significantly reduced responses,indicative of desensitization (Lefkowitz et al., 1992), while aprolonged period of rest (>5 min) was required for full responserecovery. As would be expected if T1R2+3 mediate the responses to thevarious sweet compounds (i.e., GA-2, sucrose and acesulfame-K),successive application of different tastants from this panel led to fullcross-desensitization (FIG. 6 c), while sweet tastants that did notactivate this receptor complex (e.g. glucose and cyclamate) had noeffect on the kinetics, amplitude or time course of the responses. Takentogether, these results validate T1R2+3 as a sweet taste receptor.

We propose that all T1Rs encode sweet receptors: First, they are allmembers of the same receptor family. Second, T1R1, T1R2 and T1R3 aretightly co-expressed in distinct subsets of cells. Third, data ispresented herein demonstrating that two of the three T1Rs combine tofunction as a validated sweet receptor.

Spatial Map of T1R and T2R Expression

Studying the expression of T1Rs in the context of other taste modalitiesmay provide a view of the representation of sweet taste coding at theperiphery. Recently, we showed that members of the T2R family of bittertaste receptors are rarely expressed in fungiform taste buds, but arepresent in 15-20% of the cells of all circumvallate, foliate and palatetaste buds. Given that T1Rs are also expressed in the same taste buds,we examined whether there is overlap between T1R- and T2R-expressingcells. Double-labeling experiments using mixes of T1Rs and T2R probesdemonstrated that T2Rs are not co-expressed with any of the T1R familymembers (FIG. 7, see also Adler et al., 2000). This was seen in alltaste buds, and with mixes that included as many as 20 T2Rs. The strongsegregation in the expression profile of these two receptor familiesmakes an important prediction about the logic of taste coding anddiscrimination at the taste bud level: sweet and bitter are encoded bythe activation of different cell types.

A prediction of this study is that taste buds in all taste papillaecontain sweet receptor cells and that the anatomical representation ofsweet sensitivity in the oral cavity should match the topographicdistribution of T1R receptor expression. For instance, the back of thetongue and palate contain all of the T1R2+3 expressing cells, and sothey would display high sensitivity for ligands of this receptorcombination. Conversely, the front of the tongue would respond to theT1R1+3 combination, but poorly to the repertoire specific for T1R2+3.Moreover, since the front and back of the tongue are innervated bynerves originating in different ganglia (Mistretta and Hill, 1995), weconclude that T1R2+3 sweet cells must exhibit connectivity pathways thatdiffer from those of T1R1+3 cells. Interestingly, the rat is known to bemore sensitive to sucrose applied to the back of the tongue and palate,than to stimulation of the front of the tongue (Smith and Frank, 1993).Our expression and functional studies now provide a molecularexplanation to these findings.

Experimental Procedures

Molecular Cloning of T1R3

Human T1R3 was identified in the draft sequence of BAC clone RP5-89003by homology to T1R1. A fragment of rat T1R3 was amplified from genomicDNA using degenerate PCR primers designed on the basis of the humansequence. The PCR derived probe was used to identify full-length ratT1R3 from a circumvallate cDNA library (Hoon et al., 1999) and to probemouse BAC filter arrays (Incyte Genomics and Research Genetics). Thesequences of T1R3 in Sac-taster and non-taster mouse strains (C57BL/6and 129/Sv) were determined from the genomic clones. The sequence of theentire coding region of the gene of other mouse strains that are sweetsensitive: SWR, ST, C57L, FVB/N and sweet insensitive: DBA/1Lac, DBA/2,C3H, AKR, BALB/c was determined from amplified genomic DNA (JacksonLaboratory). For SWR mice, T1R3 was also sequenced from amplifiedtaste-tissue cDNA. Amongst the 11 inbred strains, we found two tasteralleles (taster 1: C57BL/6, C57L and taster 2: SWR, ST, FVB/N) and asingle non-taster allele (DBA/1Lac, DBA/2, C3H, AKR, BALB/c, 129/Sv).Taster I and taster 2 alleles differ from each other in six amino acidpositions (P61L, C261R, R371Q, S692L, I706T, G855E; one of this G855E,was missed by (Kitagawa et al., 2001; Max et al., 2001) likely due toits inclusion in the primers used in their amplifications reactions).Non-tasters differ from taster I allele in six residues (A55T, T601,L61P, Q371R, T7061, E855G), and from taster 2 in 4 amino acid positions(A55T, T60I, R261C, L692S).

Mouse T1Rs were mapped using a mouse/hamster radiation hybrid panel(Research Genetics). Physical mapping of T1R3 involved PCR based typingof T1R3 positive BAC clones for the presence of STS-markers.

In Situ Hybridization

Tissue was obtained from adult mice. No sex-specific differences ofexpression patterns were observed. Therefore male and female animalswere used interchangeably. For foliate sections, no differences inexpression pattern were observed between the papillae. Fresh frozensections (16 μm/section) were attached to silanized slides and preparedfor in situ hybridization as described previously (Hoon et al., 1999).All in situ hybridizations were carried out at high stringency(hybridization, 5×SSC, 50% formamide, 65-72° C.; washing, 0.2×SSC, 72°C.). For single-label detection, signals were developed usingalkaline-phosphatase conjugated antibodies to digoxigenin and standardchromogenic substrates (Boehringer Mannheim). Control hybridizationswith sense probes produced no specific signals in any of the tastepapillae. Cells were counted based on the position of their nucleus aspreviously described (Boughter et al., 1997). For double-labelfluorescent detection, probes were labeled either with fluorescein orwith digoxigenin. At least 50 taste buds from at least 3 differentanimals were analyzed with any combination of probes. Analkaline-phosphatase conjugated anti-fluorescein antibody (Amersham) anda horseradish-peroxidase conjugated anti-digoxigenin antibody were usedin combination with fast-red and tyramide fluorogenic substrates(Boehringer Mannheim and New England Nuclear). Confocal images wereobtained with a Leica TSC confocal microscope using an argon-kryptonlaser; 1-2 μm optical sections were recorded to ensure that anyoverlapping signal originated from single cells.

Generation of T1R3 Transgenic Mice and Behavioral Assays

An approximately 15 kb EcoRI fragment including the 6 coding exons ofT1R3 and about 12 kb upstream of the starting ATG was isolated from aC57BL/6 BAC clone. This fragment contains the stop codon of the T1R3coding sequence but lacks much of the 3′-UTR. The sequence of the entire15 kb clone was determined from a taster and a non-taster strains. Thisfragment also contains the full sequence for a glycolipidtransferase-like gene ˜3 kb upstream of T1R3, but there are neitherexpression nor amino acid sequence differences in this gene between Sactaster (SWR) or non-taster (129/Sv) strains. In the transgenicconstruct, the bovine growth hormone polyadenylation (BGH) signal frompCDNA3.0 (Invitrogen) was ligated to the 3′-end of the T1R3 gene. Thismodification allowed PCR based genotyping of mice and permitted directcomparison of the expression of T1R3 from the transgene with that fromthe normal gene. Transgenic mice were generated by pronuclear injectionof FVB/N oocytes. Since we determined that FVB/N mice are sensitive tosweet tastants, and carry a T1R3 taster allele, transgenic founders werecrossed to 129/SvJ. F1-mice carrying the transgene were thenback-crossed to 129/SvJ. F2 mice were typed for the presence of thetransgene using the BGH tag, and for homozygosity of the endogenousnon-taster T1R3 allele using a Bsp120I restriction polymorphism betweenFVB/N and 129/SvJ (see FIG. 2 a). All four genetic groups were testedbehaviorally. Mice were weaned at 3 weeks and trained for 7-10 days todrinking from two bottles of water prior to initiating testing.

For behavioral assays, 2 or 3 mice were housed per cage; mice derivedfrom different transgenic founders (and males and females) were keptseparate to allow comparison of the raw-data. The group sizes used forassays consisted of 4 or more cages, each with a minimum of 2 animals.Mice were always assayed at the low concentrations first (Fuller, 1974).In all cases, animals were given at least 2 days of water betweenconcentration series. Each test consisted of a two-bottle choice assayover a 48 hr period; the positions of the bottles were switched after 24hr. Preference ratios were calculated by dividing the consumption of thetest solution by total intake. Data from each cage were individuallyanalyzed to prevent systematic bias. The same assay was used to analyzethe taste preferences of 129/Sv, C57BL/6 and FVB/N control mice.

Heterologous Expression of T1Rs

All receptors were cloned into a pEAK10 mammalian expression vector(Edge Biosystems, Md.). Modified HEK-293 cells (PEAK^(rapid) cells; EdgeBioSystems, MD) were grown and maintained at 37° C. in UltraCulturemedium (Bio Whittaker) supplemented with 5% fetal bovine serum, 100μg/ml gentamycin sulphate (Fisher), 1 μg/ml amphotericin B and 2 mMGlutaMax 1 (Lifetechnologies). For transfection, cells were seeded ontomatrigel coated 6-well culture plates, 24-well culture plates, or 35 mmrecording chambers. After 24 h at 37° C., cells were washed in OptiMEMmedium (Lifetechnologies) and transfected using LipofectAMINE reagent(Lifetechnologies). Transfection efficiencies were estimated byco-transfection of a GFP reporter plasmid, and were typically >70%.Activity assays were performed 36-48 h after transfection for cellstransfected in 24-well culture plates and 35 mm recording chambers;cells transfected in 6-well culture plates were grown overnight,trypsinized, transferred to 96-well culture plates, and assayed 36-48hours following re-seeding.

Calcium Imaging

Transfected cells were washed once in Hank's balanced salt solutioncontaining 1 mM sodium pyruvate and 10 mM HEPES, pH 7.4 (assay buffer),and loaded with 2 μM FURA-2 AM (Molecular Probes) for 1 h at roomtemperature. The loading solution was removed and cells in 24-wellplates were incubated with 250 μl of assay buffer (cells in 96-wellplates were incubated with 50 μl) for 1 h to allow the cleavage of theAM ester. Cells expressing T1Rs and G proteins (Offermanns and Simon,1995; Chandrashekar et al., 2000; Mody et al., 2000) in 24-well tissueculture plates were stimulated with 250 μl of a 2× tastant solution(cells in 96-well plates were stimulated with 50 μl of a 2× tastantsolution). As a control for Gα15 and Gα16-Gz signaling a set of plateswas co-transfected with mGluR1 and the μ-opioid receptor and assayed forresponses to ACPD and DAMGO.

One of two imaging stations were used to measure [Ca²⁺]i changes. Onesystem comprises of a Nikon Diaphot 200 microscope equipped with a10×/0.5 fluor objective, the TILL imaging system (T.I.L.L PhotonicsGmbH), and a cooled CCD camera. Acquisition and analysis of thesefluorescence images used TILL-Vision software. Also, an OlympusIX-70/FLA microscope equipped with a 10×/0.5 fluor objective, a variablefilter wheel (Sutter Instruments), and an intensified CCD camera (SutterInstruments) was utilized. VideoProbe software (Instrutech) was used foracquisition and analysis of these fluorescence images. Generally,individual responses were measured for 60 s. The F₃₄₀/F₃₈₀ ratio wasanalyzed to measure [Ca²⁺]i.

Kinetics of activation and deactivation were measured using a bathperfusion system. Cells were seeded onto a 150 μl microperfusionchamber, and test solutions were pressure-ejected with a picospritzerapparatus (General Valve, Inc.). Flow-rate was adjusted to ensurecomplete exchange of the bath solution within 4 s. Responses weremeasured from 80 individual responding cells.

List of Tastants

The following tastants were tested, with the following typical maximalconcentrations: sucrose (250 mM), sodium saccharin (25 mM), N-methylsaccharin (5 mM), dulcin (2 mM), aspartame (2 mM), palatinose (250 mM),sodium cyclamate (15 mM), guanidinoacetic acid-1 (1 mM), guanidinoaceticacid-2 (1 mM), guanidinoacetic acid-3 (1 mM), acesulfame-K (10 mM),glucose (250 mM), maltose (250 mM), lactose (250 mM), fructose (250 mM),galactose (250 mM), xylitol (250 mM), raffinose (250 mM), sorbitol (250mM), trehalose (250 mM), thaumatin (0.1%), monellin (0.1%), alanine (20mM), glycine (20 mM), arginine (20 mM), monosodium glutamate (20 mM),cycloheximide (5 μM), denatonium (10 mM), phenyl-thiocarbamide (2.5 mM).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1-54. (canceled)
 55. An isolated sweet taste receptor comprising a T1R3polypeptide, wherein the T1R3 polypeptide is encoded by a nucleotidesequence that hybridizes under moderately stringent hybridizationconditions to a nucleotide sequence encoding an amino acid sequence ofSEQ ID NO:15, 20, 23, or
 25. 56. The isolated receptor of claim 55,wherein the T1R3 polypeptide is encoded by a nucleotide sequence thathybridizes under highly stringent hybridization conditions to anucleotide sequence encoding an amino acid sequence of SEQ ID NO:15, 20,23, or
 25. 57. The isolated receptor of claim 55, wherein the T1R3polypeptide has an amino acid sequence of SEQ ID NO:15, 20, 23, or 25.58. The isolated receptor of claim 55, wherein the receptor comprises aT1R3 polypeptide and a heterologous polypeptide.
 59. The isolatedreceptor of claim 58, wherein the T1R3 polypeptide and the heterologouspolypeptide are non-covalently linked.
 60. The isolated receptor ofclaim 58, wherein the T1R3 polypeptide and the heterologous polypeptideare covalently linked.
 61. The isolated receptor of claim 58, whereinthe heterologous polypeptide is a T1R2 polypeptide that is encoded by anucleotide sequence that hybridizes under moderately stringenthybridization conditions to a nucleotide sequence encoding an amino acidsequence of SEQ ID NO:7, 8, or
 9. 62. The isolated receptor of claim 58,wherein the heterologous polypeptide is a T1R2 polypeptide is encoded bya nucleotide sequence that hybridizes under highly stringenthybridization conditions to a nucleotide sequence encoding an amino acidsequence of SEQ ID NO:7, 8, or
 9. 63. The isolated receptor of claim 62,wherein the T1 R2 polypeptide has an amino acid sequence of SEQ ID NO:7,8, or
 9. 64. The isolated receptor of claim 55, wherein the receptor hasG protein coupled receptor activity.
 65. The isolated receptor of claim55, wherein the receptor specifically binds to antibodies raised againstSEQ ID NO: 15, 20, 23, or
 25. 66. An isolated sweet taste receptorcomprising a T1R3 polypeptide and a T1R2 polypeptide, wherein the T1R3polypeptide is encoded by a nucleotide sequence that hybridizes underhighly stringent hybridization conditions to a nucleotide sequenceencoding an amino acid sequence of SEQ ID NO:15, 20, 23, or 25; andwherein the T1R2 polypeptide that is encoded by a nucleotide sequencethat hybridizes under highly stringent hybridization conditions to anucleotide sequence encoding an amino acid sequence of SEQ ID NO:7, 8,or
 9. 67. An antibody that specifically binds to the taste receptorclaim
 55. 68. The antibody of claim 67, wherein the antibodyspecifically binds to a taste receptor comprising T1R2 and T1 R3. 69.The antibody of claim 67, wherein the T1R2 polypeptide and the T1R3polypeptide are non-covalently linked.
 70. The antibody of claim 67,wherein the T1R2 polypeptide and the T1R3 polypeptide are covalentlylinked.
 71. An isolated polypeptide encoded by a nucleic acid thathybridizes under moderately stringent conditions to a nucleic acid thatencodes an amino acid sequence of SEQ ID NO:15, 20, 23, or
 25. 72. Thepolypeptide of claim 71, wherein the polypeptide is encoded by a nucleicacid that hybridizes under highly stringent conditions to a nucleic acidthat encodes an amino acid sequence of SEQ ID NO:15, 20, 23, or
 25. 73.The polypeptide of claim 71, wherein the polypeptide is encoded bynucleic acid that encodes an amino acid sequence of SEQ ID NO:15, 20,23, or
 25. 74. The polypeptide of claim 71, wherein the polypeptide isencoded by nucleic acid that has a nucleotide sequence of SEQ ID NO:14,19, 22, or
 24. 75. An antibody that specifically binds to thepolypeptide of claim
 71. 76. An isolated nucleic acid that hybridizesunder moderately stringent conditions to a nucleic acid that encodes anamino acid sequence of SEQ ID NO:15, 20, 23, or
 25. 77. The nucleic acidof claim 76, wherein the polypeptide is encoded by a nucleic acid thathybridizes under highly stringent conditions to a nucleic acid thatencodes an amino acid sequence of SEQ ID NO:15, 20, 23, or
 25. 78. Thenucleic of claim 76, wherein the polypeptide is encoded by nucleic acidthat encodes an amino acid sequence of SEQ ID NO:15, 20, 23, or
 25. 79.The nucleic acid of claim 76, wherein the nucleic acid that has anucleotide sequence of SEQ ID NO:14, 19, 22, or
 24. 80. An isolatedpolypeptide encoded by a nucleic acid that hybridizes under moderatelystringent conditions to a nucleic acid that encodes an amino acidsequence of SEQ ID NO:7, 8, or
 9. 81. The polypeptide of claim 80,wherein the polypeptide is encoded by a nucleic acid that hybridizesunder highly stringent conditions to a nucleic acid that encodes anamino acid sequence of SEQ ID NO:7, 8, or
 9. 82. The polypeptide ofclaim 80, wherein the polypeptide is encoded by nucleic acid thatencodes an amino acid sequence of SEQ ID NO:7, 8, or
 9. 83. Thepolypeptide of claim 80, wherein the polypeptide is encoded by nucleicacid that has a nucleotide sequence of SEQ ID NO:10, 11, or
 12. 84. Anantibody that specifically binds to the polypeptide of claim
 80. 85. Anisolated nucleic acid that hybridizes under moderately stringentconditions to a nucleic acid that encodes an amino acid sequence of SEQID NO:7, 8, or
 9. 86. The nucleic acid of claim 85, wherein thepolypeptide is encoded by a nucleic acid that hybridizes under highlystringent conditions to a nucleic acid that encodes an amino acidsequence of SEQ ID NO:7, 8, or
 9. 87. The nucleic of claim 85, whereinthe polypeptide is encoded by nucleic acid that encodes an amino acidsequence of SEQ ID NO:7, 8, or
 9. 88. The nucleic acid of claim 85,wherein the nucleic acid that has a nucleotide sequence of SEQ ID NO:10,11, or 12.